JXB Advance Access originally published online on February 27, 2004
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Journal of Experimental Botany, Vol. 55, No. 398, pp. 815-823, April 1, 2004
© 2004 Oxford University Press
Cell and Molecular Biology, Biochemistry and Molecular Physiology |
Unusual regulatory nitrate reductase activity in cotyledons of Brassica napus seedlings: enhancement of nitrate reductase activity by ammonium supply
Received 15 September 2003; Accepted 17 November 2003
1 Unité de Nutrition Azotée des Plantes, INRA Versailles, Route de Saint Cyr, F-78000 Versailles, France
2 Laboratoire de Physiologie de la Différenciation Végétale, Bât. SN2, Université des Sciences et Technologies de Lille, F-59655 Villeneuve d‘Ascq Cédex, France
* To whom any correspondence should be addressed. E-mail: christophe.vuylsteker{at}univ-lille1.fr
Abbreviations: NR, nitrate reductase; NRA, nitrate reductase activity; GS, glutamine synthetase; GOGAT, 2-oxoglutarate-glutamate-amino-transferase or glutamate synthase.
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Abstract |
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The effect of supplying either nitrate or ammonium on nitrate reductase activity (NRA) was investigated in Brassica napus seedlings. In roots, nitrate reductase activity (NRA) increased as a function of nitrate content in tissues and decreased when ammonium was the sole nitrogen source. Conversely, in the shoots (comprising the cotyledons and hypocotyl), NRA was shown to be independent of nitrate content. Moreover, when ammonium was supplied as the sole nitrogen source, NRA in the shoots was surprisingly higher than under nitrate supply and increased as a function of the tissue ammonium content. Under 15 mM of exogenous ammonium, the NRA was up to 2.5-fold higher than under nitrate supply after 6 d of culture. The NR mRNA accumulation under ammonium nutrition was 2-fold higher than under nitrate supply. The activation state of NR in shoots was especially high compared with roots: from nearly 80% under nitrate supply it reached 94% under ammonium. This high NR activation state under ammonium supply could be the consequence of the slight acidification observed in the shoot tissue. The effect of ammonium on NRA was only observed in cotyledons and when more than 3 mM ammonium was supplied. No such NRA increase was evident in the roots or in foliar discs. Addition of 1 mM nitrate under ammonium nutrition halved NRA and decreased the ammonium content in shoots. Thus, this unusual NRA was restricted to seedling cotyledons when nitrate was lacking in the nitrogen source.
Key words: Ammonium, Brassica napus, cotyledons, hypocotyl, nitrate, nitrate reductase.
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Introduction |
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Nitrate and ammonium constitute the two main soil nitrogen sources available to plants. Nitrate assimilation usually takes place in leaves and begins with the reduction of nitrate to nitrite, by nitrate reductase (NR, EC 1.6.6.1 [EC] ), in the cytosol. Nitrite is transported to the chloroplast to be reduced to ammonium by nitrite reductase (NiR, EC 1.7.7.1 [EC] ). The plastidic isoform of glutamine synthetase (GS2, EC 6.3.1.2 [EC] ) then catalyses the incorporation of ammonium into glutamate producing glutamine while the plastidic ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1 [EC] ) catalyses the transfer of the amide group from glutamine to
-oxoglutarate, thus generating two molecules of glutamate. Conversely, ammonium coming from soil absorption is rapidly assimilated in roots by the cytosolic isoform of glutamine synthetase (GS1), the NADH-GOGAT and possibly the Fd-GOGAT. Thereafter, most amino acids are generated by transamination reactions. Globally, the nitrogen assimilation cost could represent up to 25% of the photosynthetic supply (Guerrero et al., 1981). Taking into account both uptake and assimilation costs, ammonium should be preferred to nitrate as a nitrogen source. However, ammonium nutrition usually has deleterious effects on growth such as lowering of the contents of mineral cations and organic anions and depriving cells of osmotic adjustment (Salsac et al., 1987). Most steps in the nitrate assimilatory pathway are nitrate inducible. For instance, nitrate strongly stimulates the transcription of nitrate transporters (Quesada et al., 1997), nitrate reductase and nitrite reductase genes (for a review see Vedele and Caboche, 1996). By contrast, ammonium or its metabolic products exert inhibitory effects on the nitrate assimilatory pathway. Thus, nitrate inducibility of its transport is inhibited by ammonium (Zhuo et al., 1999). Glutamine and asparagine are able to inhibit nitrate uptake by the roots (Quesada et al., 1997; Filleur and Daniel-Vedele, 1999), as well as the accumulation of nitrate reductase and nitrite reductase mRNAs (Faure et al., 1991).
However, some constitutive NR isoforms have been found in soybean, corn, barley, wheat, and mustard (for a review see Solomonson and Barber, 1990) and these constitutive isoforms could fulfil specific functions independent from nitrate assimilation such as NO synthesis (Dean and Harper, 1988; Yamasaki and Sakihama, 1999). Their involvement in nitrate signalling seems unlikely as soybean mutants lacking constitutive isoforms did not show any modification of the regulation of the mRNA steady-state level of their inducible NR isoform (Santucci et al., 1995).
During its early development, rapeseed displays an embryo-specific nitrate reductase mRNA accumulation. Indeed, both zygotic and androgenic embryos were shown to express nitrate reductase genes independently of the presence of nitrate (Fukuoka et al., 1996). Under greenhouse conditions, NRA can be detected in the walls of siliques and green seeds although their nitrate contents are very low (Leleu et al., 2000). In order to verify if this nitrate-independent NR regulation is still valid after germination, seeds were submitted to various ammonium or/and nitrate regimes. The data show that nitrate was not essential to the induction of the NRA and NR expression in rapeseed cotyledons. Moreover, ammonium supply, in the absence of nitrate, stimulated the NRA in shoots more than nitrate.
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Materials and methods |
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Plant material and culture conditions
Seeds of Brassica napus L. cv. Capitol were surface-sterilized with sodium hypochlorite (65 g l–1) and germinated for 2 d in Petri dishes containing a sterile germination medium, composed of 10 g l–1 of sucrose, 5 g l–1 of agar adjusted to pH 5.8 and free of any nitrogen source. The plantlets were then transferred to tubes in sterile environment. All manipulations were done aseptically to avoid nitrification in the ammonium media by microbial contamination The mineral composition of this second medium was based on Heller’s medium (Heller, 1953), except for the nitrogen source. Nitrogen was supplied as NaNO3, NH4Cl or a mixture of these two salts. No more sucrose was added. Five grams of agar per litre were added. The presence of 0.5 g l–1 of MES allowed the pH of the media to be buffered around 5.8±0.1 during the whole growth period independently of the form of N supplied. No trace of nitrate, using Cataldo’s method described below (Cataldo et al., 1975), could be detected in the media containing NH4Cl as the sole nitrogen source during the whole growth of the plants.
Plants were kept in the culture room and were submitted to a 16/8 h photoperiod. The photon flux measured was 100 µmol m–2 s–1 and the temperature was 27/22 °C (day/night) at plantlet level. Plants were sampled 8 h after the beginning of the day cycle when the nitrate reductase activity was at its maximum. Shoots comprising the two cotyledons and the hypocotyl were separated from the roots.
The leaves used in the foliar discs experiment were taken from 20-d-old plantlets that had been grown on a low nitrate medium (3.5 mM NaNO3) necessary for leaf development. After 2 weeks of nitrate starvation, no more nitrate was detectable in the leaves, as assayed by Cataldo’s method (Cataldo et al., 1975). The cotyledons came from 3-d-old seedlings, grown on nitrogen-free germination medium. Leaf discs, with a diameter of 10 mm, or cotyledons were cut out and floated on Petri dishes containing the liquid medium (10 mM MES–KOH pH7, 40 mM KCl, 10 mM CaCl2) whose osmotic pressure had been adjusted to that of the tissues by adding PEG 400. The Petri dishes were gently stirred and kept for 48 h under the same culture conditions as those used for seedling growth.
All tissue samples were frozen separately in liquid nitrogen before being ground in liquid nitrogen, and stored at –80 °C until extraction.
Determination of nitrate and ammonium contents
Five hundred mg of fresh matter were added to 1 ml of deionized water and shaken for 1 h at 45 °C. Samples were centrifuged at 15 000 g for 20 min.
Nitrate content was determined according to Cataldo et al. (1975). Fifty microlitres of supernatant were added to 200 µl of 5% (w/v) salicylic acid in concentrated H2SO4 97% (w/v). After 15 min of incubation at room temperature, 1 ml of 4 N NaOH was added. When the solution came to room temperature, the optical density was measured at 410 nm. Nitrate content was then calculated according to a standard curve. The nitrate content was expressed as µmol NO3– g–1 FW.
Ammonium content was determined on 50 µl of the supernatant using 1 ml of Nessler reagent (Merck). Optical density was measured at 404 nm. The NH4+ content was determined by using a standard curve and expressed as µmol NH4+ g–1 FW.
pH determination
According to Kaiser and Brendle-Behnisch (1995), 320 mg of fresh matter was added to 640 µl of deionized water. The suspension was centrifuged at 15 000 g for 10 min, and the pH of the supernatant was measured with an E50 WTW type glass electrode.
Chlorophyll content
Shoots were harvested and ground in 80% acetone (v/v). The suspension was vacuum filtered. After centrifugation (5000 g for 10 min) of the filtrate, total chlorophyll content was measured by spectrophotometer on the supernatant according to Bruinsma (1963).
Soluble protein extraction
Frozen material (0.5 g) was ground in a mortar and homogenized with 4 ml of 50 mM HEPES–KOH (pH 7.5) buffer, containing 0.5 mM EDTA, 5.5 mM MgCl2, 14 mM ß-mercaptoethanol, 0.1% Triton X100 (v/v), 10% glycerol (v/v), 10% polyvinyl-pyrrollidone (w/v), 50 µM leupeptin, and 0.5 mM PMSF. After centrifugation at 5000 g for 20 min at 4 °C, the supernatant was assayed for NR and GS activities.
NR activity
Nitrate reductase activity (NRA) was assayed in 400 µl of 50 mM HEPES–KOH (pH 7.5) buffer containing 10 mM KNO3, 2 mM EDTA, 0.2 mM NADH, and 10 µM FAD. The reaction was started after addition of 100 µl of the protein extract. Incubation was carried out at 30 °C for 5 min. The reaction was stopped by adding 50 µl of 0.5 M zinc acetate. Excess NADH was destroyed by adding 20 µl of 300 µM phenazine methosulphate during 15 min of incubation. Nitrite was revealed at 540 nm by adding 350 µl of sulphanilamide 0.673 M in 3 M HCl and 350 µl of 29.7 mM N'-naphthyl-ethylene-diamine-dihydrochloride. NRA was expressed as nmol NO2– min–1 g–1 FW. No NADPH NRA was detected in either shoot or root extracts. Activation state was estimated by calculating the ratio of NRA under Mg2+ conditions to NRA measured under EDTA conditions as described above. Under Mg2+-NRA conditions, 5 mM MgCl2 were added to the incubation buffer instead of 2 mM EDTA. For each measurement, one blank was made by adding zinc acetate prior to protein extract.
ELISA immunoquantification of NR protein content
Soluble proteins were extracted under non-denaturing conditions by adding 8 ml of a 50 mM HEPES-KOH (pH 8.2) buffer containing 0.1 M NaCl, 1 mM EDTA, 5 mM cysteine, 1 µM leupeptin, and 5 µM FAD to 1 g of FW. NR proteins were then precipitated with 45% (NH4)2SO4. The precipitate was subsequently dissolved in 200 µl of the extraction buffer described above. The NR protein content was quantified by the two sites ELISA procedure using a monoclonal mouse antibody directed against the maize NR and a rabbit polyclonal antibody directed against the spinach NR. Detection was performed by using goat antibodies directed against IgG coupled to alkaline phosphatase from Sigma. Both antibodies were previously successfully tested against rapeseed NR by western blot analysis and immunoprecipitation followed by NRA assay.
Total RNA extraction and northern blot analysis
Total RNA was extracted from tissues using the Tri Reagent Mix® extraction kit (Euromedex, France). The amount of total RNA was 50 µg for the NR blot analysis. Total RNA was electrophoretically fractioned in a 1.5% (w/v) agarose formaldehyde denaturing gel. Subsequently, blotting was carried out on Hybond-N+ membranes (Amersham). DNA probes were labelled with [-32P]dCTP (ICN) using random priming (T7 Quickprime, Pharmacia) and hybridized according to Church and Gilbert (1981). The NR probe used was the AtNia2 cDNA from Arabidopsis thaliana (GenBank accession number NM_103364
[GenBank]
). The 18S rRNA probe was a PCR amplified fragment issued from Cichorium intybus. Membranes were exposed to X-ray films (Kodak) at –80 °C. The intensity of the hybridization signals was measured after digitalization. Relative accumulation of mRNAs was estimated by calculating the ratios of NR mRNA signal to 18S rRNA signals.
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Results |
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When nitrate was the sole nitrogen source, the best growth was achieved when it was supplied at 15 mM. This level of nitrogen was used in the first experiments when nitrogen was provided as a mix of nitrate and ammonium at different ratios.
In seedlings grown for 7 d when NH4+ was the sole nitrogen source, nitrate was not detected either in the shoots, comprising hypocotyl and cotyledons, or in the roots (Fig. 1A). This ruled out any significant nitrate release from internal stores. When nitrate was applied, the nitrate content was always higher in shoots than in roots. It increased linearly in both parts as the nitrate concentration in the medium increased. When nitrate was the sole nitrogen source, the shoots and roots accumulated more nitrate than could be expected regarding the nitrate content of other NO3–/NH4+ ratios (Fig. 1A). Except when NH4+ was the sole nitrogen source, the ammonium contents of shoots and roots were not significantly different and increased as ammonium was supplied in greater proportion (Fig. 1B). When NH4+ was the sole nitrogen source, a very large increase in the ammonium content occurred only in the shoots (Fig 1B).
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Maximum NRA in roots was observed in the presence of nitrate alone. However, under NH4+ supply, little NRA remained in the roots (Fig. 1C). Regardless of the nitrate content, NRA in the shoots remained stable and higher than in roots (Fig. 1C). Contrary to all expectations, when only ammonium was supplied, NRA in the shoot increased about 2.5-fold, although no nitrate was detectable in tissues (Fig. 1A, C). When NRA was expressed as a function of soluble protein content instead of fresh weight, NRA was still 2-fold higher under ammonium supply. Soluble proteins accumulation under ammonium supply could not alone explain the NRA increase.
The growth and NRA were monitored over 9 d under two contrasting N regimes consisting of 15 mM of nitrogen supplied either as NaNO3 or NH4Cl (Fig. 2). From the fourth day on, the fresh weight of the shoots greatly increased under nitrate supply. No such increase was observed under ammonium (Fig. 2A, B). From the first up to the fourth day of culture, the plantlets were similar whether grown on nitrate or ammonium (Fig. 2A). At the sixth day, the growth of plantlets was delayed under ammonium nutrition. After 9 d under ammonium supply, the cotyledons became slightly curly and chlorotic (Fig. 2A). The chlorophyll content was about 20% lower under ammonium nutrition (Table 1). The shoot pH was lowered by 0.6 units under ammonium nutrition compared to nitrate one (Table 1).
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NRA of shoots of nitrate-fed plants increased from the first day on to the fourth and then slightly decreased (Fig. 2C). In shoots of NH4+-fed plants, NRA was 3-fold higher than under nitrate supply as early as the first day. Thereafter, NRA still increased in shoots of ammonium-fed plants remaining at least 2-fold higher than in shoots of nitrate-fed plants until the sixth day before decreasing. The NRA in roots was lower, comparatively, than shoots in both nitrogen nutritions, but was very low in roots from plants under ammonium supply. As NRA in the shoots reached a maximum on the sixth day of growth under ammonium nutrition, further experiments were carried out on 6-d-old plantlets.
Plantlets were grown for 6 d, in the absence of nitrate, in a range of ammonium concentrations from 1–20 mM to test the ammonium dose effect upon the NRA. The ammonium content in shoots showed a sharp increase when the ammonium concentration reached 10 mM in the nutrient medium (Fig. 3). The NRA in shoots increased with the ammonium content. The NRA was the highest at 15 mM of exogenous ammonium supply or 8.2 µmol g–1 FW tissular ammonium. At higher ammonium supply, the NRA dropped but remained higher than under 15 mM nitrate supply.
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In order to test whether the NRA in leaves responded to the presence of ammonium, as in seedling shoots, leaf discs were used from plants grown on an N-limited medium (3.5 mM NO3–). After 2 weeks of nitrate starvation, the leaves contained no more nitrate (data not shown). In contrast to cotyledons, NRA in foliar discs was slightly decreased by ammonium supply after 1 d of incubation (Fig. 4). However, the NRA in excised cotyledons obtained from 3-d-old seedlings increased 2-fold in the liquid medium containing 15 mM NH4Cl compared with the 15 mM NaNO3 supply (Fig. 4).
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The effect of ammonium or nitrate additions on NRA of plantlets grown, respectively, under nitrate or ammonium supply was examined. When 1 mM NH4Cl was added under nitrate supply for 1 d, the NRA was lowered by about 20% and the ammonium content increased 4-fold. Conversely, when 1 mM NaNO3 was added to the medium of ammonium supply, the NRA of shoots was reduced by half and the ammonium content by a third (Table 2).
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Accumulation of nitrate reductase mRNA was investigated by northern blot analysis. From the second day on, the ratio of the hybridization signals of NR genes to 18S was higher in the shoots of ammonium-fed plants than in nitrate-fed ones (Fig. 5). Then, the NR mRNA accumulation decreased under both N-regimess. However, NR mRNA levels remained 2-fold higher under ammonium supply.
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The NR protein content was measured by ELISA immunoquantification. After 6 d, the shoots of ammonium-fed plantlets contained 7-fold more NR protein than the nitrate-fed ones (Table 3). NRA was measured either in the presence of 5 mM Mg2+ (Mg-NRA) or 2 mM EDTA (EDTA-NRA). The presence of Mg2+ in both the extraction and incubation buffers maintains coupling between inhibitory 14-3-3 proteins and phospho-NR proteins. Conversely, EDTA prevents such interaction and thus EDTA-NRA represents the potential contribution of both the phospho- and dephospho-NR proteins independently of the presence of the inhibitory protein (Moorhead et al., 1999). The NR activation state in shoots was higher than in roots (Table 3). Under ammonium supply, although the NR activation state in roots was lowered by a third, it reached up to 94% in shoots compared with 80% under nitrate supply (Table 3). Ammonium itself had no effect on NRA activation state when it was added directly to the incubation medium of protein extracts from shoots of nitrate-fed plantlets (data not shown). The specific NRA was expressed as the ratio of EDTA-NR activity to the NR protein content. This specific NRA under ammonium nutrition was only 44% of the one measured under nitrate supply (Table 3).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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When nitrate was supplied with ammonium to rapeseed seedlings, nitrate content in both roots and shoots (comprising hypocotyl and cotyledons) increased linearly as a function of the amount of nitrate supplied. However, when NO3– was the sole nitrogen source, nitrate content was higher than expected. Inhibition of nitrate uptake by ammonium could explain the great increase in nitrate accumulation when ammonium was omitted (Zhuo et al., 1999). The amount of ammonium also increased linearly as a function of the ammonium present in the medium. When ammonium was the sole nitrogen source, the ammonium content increased considerably in the shoots. This ammonium accumulation level under ammonium supply was very high compared with the nitrate accumulation level under nitrate supply (10 µmol NH4+ g–1 FW versus 4 µmol NO3– g–1 FW). When ammonium was supplied at more than 10 mM, the ammonium content increased greatly. This high ammonium accumulation may not be due to higher ammonium transport. Indeed, Kronzucker et al. (1996) showed that ammonium accumulation inhibits its transport. Such accumulation of ammonium could be due to its lower assimilation. The impact of supplying high amounts of ammonium or glutamine on the GS-GOGAT cycle is currently being investigated.
The mRNA accumulation was about two times under ammonium nutrition than under nitrate. It is possible that a specific NR isoform could possibly account for the ammonium induced NRA in rapeseed shoots. Alternatively, some modification by ammonium in the amounts of metabolites acting as regulators of NR expression supply may have induced NR expression. It should be noticed that Fukuoka et al. (1996) have shown that both NR genes identified in rapeseed presented a nitrate-independent expression in embryos. The BnNRA and BnNRB cDNA sequences of rapeseed (GenBank accession numbers D38219 [GenBank] and D38220 [GenBank] ) were very similar to each other (87% of identity) and their lengths were equivalent (2990 versus 2920 nucleotides). BnNRA and BnNRB were also quite similar to AtNia2 with, respectively, 72% and 76% of identity. It could be expected that the NR signal measured represents the accumulation of both NR rapeseed mRNAs. So, even if the expression of the different NR isoforms was not investigated separately, it can be assumed that expression of both genes was nitrate-independent in rapeseed cotyledons. However, it was not possible to distinguish between a transcriptional effect and an increased lifetime of a transcript. Polyamines that could be synthesized under ammonium nutrition could regulate the mRNA stability (Veress et al., 2000). However, Cannons and Pendleton (1994) reported that NR mRNA stability could be decreased in the presence of ammonium in Chlorella vulgaris.
Effects on post-translational regulation were also shown. These data show the very high activation state of NR in shoots, whatever the nitrogen source supplied. In the presence of ammonium instead of nitrate, it increased from 80% to 94%. No such high activation state could be shown in roots where ammonium supply decreased the activation state. The high activation state in shoots could contribute to the modification of NR protein turnover. Indeed, it has been proposed that NR phosphorylation, and subsequent binding to 14-3-3 proteins could be a prerequisite for NR degradation (for review see Kaiser and Huber, 2001). Slight acidification was observed in shoots when ammonium was supplied at 15 mM. This pH decrease probably reflects the usual proton-producing process of long-term ammonium assimilation (see Gerendas and Ratcliffe, 2000, for a discussion). According to Kaiser and Brendle-Behmish’s experiments (Kaiser and Brendle-Behmish, 1995) on excised spinach leaves, the decrease in pH could stimulate NRA probably by modifying the phosphorylation status of the NR protein. However, in the experiments from this study, acidification due to ammonium assimilation did not act on the NRA of roots as in the shoots under ammonium supply. The lower activation state observed for NR in roots may be due to more efficient buffering of the acidification due to ammonium assimilation by MES in these organs. It may be inferred, therefore, that the pH effect of ammonium treatment probably explains the higher activation state of NR in shoots under ammonium nutrition, but does not constitute the main mechanism by which NRA of shoots increased. Indeed, even if this acidification could enhance the life-time of NR protein by lowering the NR phosphorylation rate and, consecutively, its degradation, this can not explain the existence of NRA in the absence of nitrate induction. Saroop et al. (2000) showed that the nitrate is not required when NR is induced by light treatment in mustard (Brassica juncea). This light-induced NR had very low turnover compared with nitrate-induced NR. Moreover, Mehta and Srivastava (1980), showed that ammonium induces NRA only when maize leaves were light- incubated. The turnover of this ammonium-induced and light-induced NR was lower than in the nitrate-induced one. These examples may account for the same nitrate-independent NR under ammonium and light treatments that were observed in the present experiments. Furthermore, Man et al. (1999) showed that the NR activation state of barley leaves was higher when nitrate supply was low compared with a high nitrate supply. Thus, there is some evidence that nitrate-independent NR is differently regulated at the post-translational level. This could explain the high activation state measured in the experiments from this study on both nitrogen sources.
The specific NRA, calculated as the ratio of EDTA-NRA to NR protein content, under ammonium nutrition was less than half of the NRA under nitrate nutrition. The non-functionality of the NR proteins accumulated in shoots deprived of the nitrate substrate could also be a determining factor responsible for a slower turnover of NR protein. Nussaume et al. (1995) have shown that the constitutive expression of a mutant form of NR missing 56 amino acids in a region preceding the MoCo domain in tobacco plants prevented the NR protein level drop even 48 h after the plants were transferred into darkness. This contrasts with the previous work carried out by Vincentz and Caboche (1991) who showed that constitutive expression of functional NR in tobacco plants did not prevent the decrease in NR protein content after transfer into darkness. These two experiments allowed Crawford (1995) to propose that the structural integrity of the NR enzyme could be a determinant factor in NR turnover. Interestingly, in Chlorella vulgaris, Solomonson and Barber (1990) have reported that ammonium as the sole nitrogen source induces cyanide production that inhibits NRA by binding to the molybdenum cofactor and by stopping the electron flux. However, such cyanide inhibition of NRA has not been shown in higher plants.
When considering NR activity, the response to nitrate and ammonium supplies was quite usual for rapeseed roots. Under mixed nitrogen supply, their NRA increased with nitrate and maximum activity was found under nitrate supply. By contrast, the NRA in shoots was surprising. First, under mixed nitrogen regimes, the NRA was poorly affected in shoots by the nitrate and ammonium contents. Moreover, maximum NRA was measured when ammonium was the sole source of nitrogen. This high NRA was supported by higher NR mRNA and NR protein levels and a lower sensitivity to magnesium inhibition of its activity. This response under ammonium supply is dependent on the ammonium content in shoots, provided that no nitrate was added. It was quite reversible by low nitrate supply. If ammonium stimulated the NRA in shoots of entire plantlets as well as in excised cotyledons, it could not act in foliar discs. Thus the organ specificity of cotyledons is essential to the ammonium stimulation of NRA.
Inhibitory effects of ammonium or reduced nitrogen metabolites such as glutamine were commonly observed in plants or organs in the presence of nitrate. Thus, the results from this study should be more pertinently compared with experiments performed on Clematis vitalba (Bungard et al., 1999) or Quercus rubra (Truax et al., 1994), which showed that NRA in leaves was significantly higher in the presence of ammonium as a sole nitrogen source than in the presence of nitrate. Although with western blot rather than ELISA, Bungard et al. (1999) did not show that protein levels of nitrate-assimilating enzymes were increased along with NRA under ammonium nutrition. It has been shown here that, in rapeseed, ammonium acted on both mRNA accumulation and NR post-translational regulations.
Fukuoka et al. (1996) suggest that cytokinin might play a role in the nitrate-independent regulation of NR during embryo development in rapeseed. They mentioned the experiments of Kende and Zeevaart (1997) who showed that benzyladenine could induce NRA in Agrostemma githago embryos in the absence of nitrate. Since then, Sood et al. (2000) have reported the existence of an ammonium-induced NR isoform in radish (Raphanus sativus) cotyledons promoted by kinetin treatment. Takei et al. (2002) reported that ammonium could increase cytokinin translocation to leaves. This accumulation of cytokinin could lead to NRA induction (Sakakibara, 2003). Faure et al. (1994) have shown a link between carbohydrate and nitrogen metabolism and cytokinin action in Nicotiana plumbaginifolia cotyledons. This NR expression without nitrate induction could be organ or developmental dependent. For example, the rbcS-1A (chlorophyll a/b-binding, cab) gene expression has been reported by Brusslan and Tobin (1992) to be developmental-dependent and occurred in Arabidopsis cotyledons without external stimuli, by contrast with later in the development, when this gene expression becomes light-dependent. Such stimuli- independent gene regulation could also occur in the case of the NR expression in the rapeseed cotyledons in the absence of nitrate.
If such a nitrate-independent NR isoform could be specifically induced during rapeseed embryo development, the function of this nitrate-independent isoform remains to be elucidated. Could NO synthesis represent an important alternative role for constitutive NR (Yamasaki and Sakihama, 1999) thus justifying some specific regulation features involved in early physiological or developmental processes?
Alternatively, this unusual NR response could result from the concomitant presence of particular developmental and metabolic contexts occurring in seedling cotyledons due to the mobilization of stored protein and lipid, the unusual absence of nitrate, and the presence of large amounts of ammonium in the nitrogen supply. Standard regulation of NRA occurred in rapeseed plantlets in roots and leaves and when ammonium amounts in the medium was less than 3 mM. This unusual NRA regulation restricted to cotyledons may highlight a particular N/C metabolic coordination. The analysis of the impact of the assimilation of large amounts of ammonium in the absence of nitrate and of lipid mobilization on nitrogen metabolism could help to understand this odd NRA regulation.
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Acknowledgements |
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We wish to thank HN Truong-Cellier and C Meyer for helpful discussion. We thank Dr G Congero (INRA, Montpellier) for the gift of the anti-NR maize monoclonal antibody polyclonal and Dr T Moureaux (INRA, Versailles) for the gift of the anti-NR maize polyclonal antibody. We thank F Vedele for the NR cDNA probe. This work was supported by grants from Conseil Regional Nord-Pas de Calais.
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References |
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