Journal of Experimental Botany, Vol. 53, No. 370, pp. 875-882,
April 15, 2002
© 2002 Oxford University Press
Original Papers |
Modulation of nitrate reductase: some new insights, an unusual case and a potentially important side reaction
1 Julius-von-Sachs-Institut für Biowissenschaften, Julius-von-Sachs-Platz 2, D-97082 Würzburg, Germany
2 University of Bielefeld, Lehrstuhl für Stoffwechselphysiologie und Biochemie der Pflanzen, Universitätsstr. 25, D-33615 Bielefeld, Germany
3 FZ Jülich, Institut für Chemie der belasteten Atmosphäre, D-52425 Jülich, Germany
Received 18 July 2001; Accepted 20 November 2001
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Abstract |
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 Top Abstract IntroductionPost-translational NR...Some new insights into...Many external factors trigger...Other ways to inactivate...Are NR modulation and...An unusual case: NR...Potentially important side...References |
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The mechanism of the post-translational modulation of nitrate reductase activity (NR, EC 1.6.6.1) is briefly summarized, and it is shown that by this mechanism nitric oxide production through NR is also rapidly modulated. New and partly unexpected details on the modulation mechanism have been obtained by using immunological techniques. The phosphorylation state of NR has been assessed with peptide antibodies raised against the serine phosphorylation motive of spinach NR. By co-immunoprecipitation experiments, 14-3-3 binding to phospho-NR and the function of Mg2+ in that process has been elucidated. Conflicting data on the role of NR phosphorylation and 14-3-3 binding in controlling NR proteolysis are discussed. A possible role of other NR inactivating proteins is also briefly considered and the regulation of NR of Ricinus communis is described as an interesting special case that differs from the ‘normal’ mechanism in several important aspects.
Key words: Cytosolic pH, nitrate reductase, nitric oxide, protein phosphorylation, 14-3-3 proteins, phosphorylation state, sugar signalling.
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Introduction |
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TopAbstractIntroductionPost-translational NR...Some new insights into...Many external factors trigger...Other ways to inactivate...Are NR modulation and...An unusual case: NR...Potentially important side...References |
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Assimilatory nitrate reductase (NR) of higher plants is highly regulated in a very complex manner, and many recent reviews have highlighted different aspects of that regulation. Here, current knowledge of the post-translational regulation is briefly summarized, putting emphasis on new insights obtained recently by immunological techniques. The function of other NR-inactivating proteins is also discussed and an example of a higher plant NR which appears to be regulated differently from the normal pattern is described in more detail. It is further demonstrated that the modulation of NR activity also modulates NO-production from nitrite and NAD(P)H.
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Post-translational NR modulation: a brief summary |
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TopAbstractIntroductionPost-translational NR...Some new insights into...Many external factors trigger...Other ways to inactivate...Are NR modulation and...An unusual case: NR...Potentially important side...References |
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Nitrate reductase (NR, EC 1.6.6.1) catalyses the transfer of two electrons from NAD(P)H to nitrate to produce nitrite. It may also catalyse a one electron transfer from NAD(P)H to nitrite to produce nitric oxide (NO) (Dean and Harper, 1988
; Yamasaki et al., 1999) and from NAD(P)H to O2 to produce superoxide anion (Barber and Kay, 1996; Yamasaki and Sakihama, 2000). It is believed that the potential toxicity of all these reaction products is the reason for the development of a complex and redundant control of NR at the transcriptional and the post-translational level. Current knowledge about NR phosphorylation and 14-3-3 binding has been recently reviewed (Kaiser and Huber, 2001; MacKintosh and Meek, 2001): Briefly, NR can be phosphorylated on a serine residue (serine 543 in spinach; Bachmann et al., 1996a) in the hinge 1 region, creating a binding site for 14-3-3 proteins (R-X-X-pS/pT-X-P). In the presence of mM concentrations of free Mg2+, binding of 14-3-3 to P-NR inactivates P-NR completely without changing kinetic constants. In the absence of divalent cations (+EDTA), all NR-forms remain fully active. Thus, comparative activity measurements with excess Mg2+ or EDTA provide a simple method to estimate the ratio of total NR activity (+EDTA=100%) and ‘real’ NR activity (+Mg2+=x%), which is defined as the ‘activation state’ of NR. Phosphorylation is catalysed by protein kinases, two of which belong to the CDPK-family (calcium-dependent protein kinases) and one is a SNF1 (sucrose non-fermenting 1)-related kinase. Phosphate (from 
-32P-ATP) is incorporated into NR in a 1:1 stoichiometry (Weiner and Kaiser, 2001). Dephosphorylation is most likely catalysed by a PP2A (protein phosphatase 2A). Binding of 14-3-3s to P-NR appears to slow down dephosphorylation (Bachmann et al., 1996b). The activity of protein kinases and phosphatases creates a dynamic equilibrium between NR and P-NR, which can be rapidly shifted by external conditions. |
Some new insights into NR phosphorylation and 14-3-3 binding using immunological techniques |
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TopAbstractIntroductionPost-translational NR...Some new insights into...Many external factors trigger...Other ways to inactivate...Are NR modulation and...An unusual case: NR...Potentially important side...References |
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Immunological techniques have provided some new and partly unexpected insights into some details of the activation/inactivation mechanism (Weiner and Kaiser, 1999
, 2000, 2001). Peptide-antibodies were raised that select for either serine 543 phospho- or dephospho-NR (spinach). The dephospho-specific antibodies blocked NR phosphorylation on ser 543. The phospho-specific antibodies prevented NR binding to 14-3-3s, but binding of the antibody to P-NR did not inactivate NR. Dephosphorylation of P-NR was prevented by these antibodies. - (1) With the aid of these antibodies, the amount of different NR-forms in extracts from illuminated or darkened spinach leaves was determined. As expected from activity measurements of NR, in the light, 1/3 of NR was already phosphorylated on ser 543. However, surprisingly little 14-3-3 was bound to that P-NR, as indicated by co-immunoprecipitation of 14-3-3s with P-NR. According to current knowledge, activity of P-NR and of free NR are almost identical, whereas P-NR–14-3-3 has practically no activity. If in extracts from illuminated leaves only a little 14-3-3 is bound to P-NR, activity should be higher than measured. The reason for that discrepancy is not known.
- (2) In the dark, NR became almost fully phosphorylated and bound to 14-3-3s. Thus, either the binding properties of 14-3-3s to P-NR are different in dark or light, or the amount of free 14-3-3s available for binding is different in illuminated or darkened leaves. Indeed, 14-3-3s themselves may be post-translationally modified, changing their binding properties (MacKintosh and Meek, 2001
). Potentially, such modifications might also change cross-reactivity with anti-14-3-3 antibodies, which might than explain the discrepancy described in (2).
- (3) In extracts from darkened leaves, two 14-3-3 dimers were pulled down together with one p-NR dimer. Thus, phosphorylation creates one serine phosphorylation site per NR monomer which binds to one 14-3-3 dimer. But there is some evidence that other sites than ser 543 may be involved in 14-3-3 binding (summarized by MacKintosh and Meek, 2001
), which would also complicate interpretation of co-immunoprecipitation data.
- (4) It has long been known that the inactivation of NR requires millimolar concentrations of free Mg2+. Theoretically, there may be two reasons: either, divalents are necessary for 14-3-3 binding, or they are required to convert the P-NR–14-3-3 complex into an inactive form. Originally it was thought that Mg2+ was a prerequisite for binding of 14-3-3s to P-NR (Kaiser and Spill, 1991
; Athwal et al., 1998). This view is supported by previous findings that co-purification of NR and 14-3-3 using Blue Sepharose required the presence of divalent cations (Provan et al., 2000). However, co-immunoprecipitation of 14-3-3s with NR showed surprisingly little difference in the amount of 14-3-3s pulled down together with NR whether Mg2+ was present or not. Also, interaction of NR and 14-3-3s in overlay experiments did not require Mg2+ (Cotelle et al., 2000). According to that, Mg2+ would only be required for inactivation of the P-NR–14-3-3 complex (Fig. 1). Free NR and dephospho-NR are both much less sensitive to Mg2+ (50% inhibition at 20 mM) than the P-NR–14-3-3 complex (50% inhibition at 
0.5 mM). Thus, while the requirement of NR modulation for millimolar concentrations of Mg2+ is beyond doubt, the exact mechanism of the action of divalent cations is not yet completely clear. To complicate things even more, NR from Ricinus communis appears different from other higher plant NRs in as much as it appears always extremely Mg2+-sensitive even in the absence of 14-3-3s (see below), and at least for NR from squash leaves it was shown that complete removal of substrates (NADH and nitrate) may convert NR into a form with increased sensitivity to inhibition by divalent cations (Lillo, 1993).
- (2) In the dark, NR became almost fully phosphorylated and bound to 14-3-3s. Thus, either the binding properties of 14-3-3s to P-NR are different in dark or light, or the amount of free 14-3-3s available for binding is different in illuminated or darkened leaves. Indeed, 14-3-3s themselves may be post-translationally modified, changing their binding properties (MacKintosh and Meek, 2001
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Many external factors trigger the modulation of NR, but what are the signalling compounds inside the cell? |
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TopAbstractIntroductionPost-translational NR...Some new insights into...Many external factors trigger...Other ways to inactivate...Are NR modulation and...An unusual case: NR...Potentially important side...References |
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While nitrate is the most important external (and internal) factor controlling NR mRNA synthesis, it appears not to have a direct effect on the NR phosphorylation state (or activation state). For example, in barley leaves grown under short-day conditions with low or high nitrate supply it was found that, although nitrate content, total NR activity and NR protein content were very different under the two conditions, the NR activation state and the degree of light/dark modulation was almost identical (Man et al., 1999
). On the other hand, under long-day conditions and with low nitrate supply, the resulting low NR activity was much less inactivated in the dark. A possible interpretation would be that this small inactivation served partly to compensate for the low NR protein content. The idea is supported by the observation that mutants with decreased NR protein content did not dark-inactivate NR as strongly and consistently as wild-type plants (Scheible et al., 1997).
While nitrate itself appears not to be directly involved in the modulation of NR activity, many other external factors are known to affect NR activation. They have been recently reviewed (Kaiser and Huber, 2001), and only some brief comments are given here. In leaves, photosynthesis activates NR. In the dark, or in the light in CO2-free air, a larger part of NR is in the inhibited form. Sugar feeding in the dark activates NR, whereas in a maize mutant lacking Calvin cycle activity light activation of NR was absent (Provan and Lillo, 1999). Remarkably, sugars (or assimilates) are also involved in controlling NR transcription (Cheng et al., 1992). Both the light activation and the activation by sugar feeding may actually be mediated by increased sugar phosphate levels, since sugar phosphates inhibit NR kinases in vitro (Bachmann et al., 1995; Kaiser and Huber, 2001). In that context it is noteworthy that during a diurnal cycle, NR activity in leaves is often decreasing during the late light phase, although sugar levels in the leaves are still increasing or at least not decreasing. This ‘afternoon depression’ of NR may reflect NR degradation (Man et al., 1999) and/or a block in NR synthesis, and is usually paralleled by decreasing nitrate concentrations in the leaves. Indeed, NR mRNA levels usually peak in the first half of the light phase and decrease thereafter (Abd-El Baki et al., 2000).
Inhibition of dark respiration by anoxia or by chemicals increases NR activity to the light level. Unfortunately, under these conditions, ratios of NR, P-NR and P-NR–14-3-3 have not yet been determined by the specific antibodies applied above. Cellular acidification or treatment with Mg2+/Ca2+-ionophores (in the presence of external EDTA) also reversibly activate NR. Whether the acidification effect is directly mediated via a pH-dependent change in the relative activities of NR-kinases or pNR-phosphatase, or whether acidification and ionophore treatment affect 14-3-3 binding or whether they affect in some unknown way concentrations of metabolic triggers (e.g. sugar levels) is not known as yet, since, for example, sugar-and sugar-P levels have not been followed systematically under all these conditions (see below). It is therefore as yet unclear whether the actual trigger is always the cytosolic sugar (or sugar-P)-concentration, or whether physical conditions like cytosolic pH may directly affect the ratios of different NR forms or 14-3-3 binding.
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Other ways to inactivate NR: NR inhibitor proteins |
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TopAbstractIntroductionPost-translational NR...Some new insights into...Many external factors trigger...Other ways to inactivate...Are NR modulation and...An unusual case: NR...Potentially important side...References |
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In contrast to 14-3-3s that quickly but reversibly inactivate NR, there are other types of proteins that inactivate NR in a different way. A protein (NRI) has been purified from spinach which causes an irreversible inhibition of NR in vitro. Experiments using artificial electron donors (reduced methyl viologen or FMN) and acceptors (NADH-ferricyanide reductase and NADH-cytocrome c reductase) indicated that the diaphorase component of the NADH–NR complex is the site of action of NRI (Sasaki et al., 1995
). The amino acid sequence deduced from NRI cDNA of spinach had a high homology to a nucleotide pyrophosphatase precursor protein of rice, and to three types of nucleotide pyrophosphatase-like proteins of Arabidopsis (Sonoda et al., 2000). It is known that nucleotide pyrophosphatases from potato catalyse the hydrolysis of pyrophosphate linkages in, for example, NADH, ATP, FAD, and others. However, other enzymes requiring NADH as coenzyme (e.g. glutamate dehydrogenase and lactate dehydrogenase) were not affected by the NRI (Sasaki et al., 1995). Thus, the effect appears to be NR specific. The electrophoretic mobility (on non-denaturating gels) of the inactivated NR was slower after incubation of NR with NRI, indicating that no partial proteolysis had taken place. On the other hand, mobility of NRI did not change (Sasaki et al., 1995). These data suggest that (i) NRI may act on FAD bound to NR, and that (ii) it mediates assembly of NR into oligomeric forms. It is not yet clear where NRI is located in the cell and what physiological role this protein has in vivo. NR mRNA increased for 2 d after cultured spinach cell were transferred into new medium and after that NR mRNA decreased. During that latter phase, NRI-mRNA levels changed inversely to NR mRNA (Sonoda et al., 2000). Therefore it was tentatively suggested that NRI may play a role in NR inactivation, for example, during senescence. However, it cannot be excluded that in vivo NiR never comes into contact with NR and that the inactivation may thus represent an artefact. |
Are NR modulation and NR turnover somehow related? |
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TopAbstractIntroductionPost-translational NR...Some new insights into...Many external factors trigger...Other ways to inactivate...Are NR modulation and...An unusual case: NR...Potentially important side...References |
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Basically, efficient regulation of an enzyme activity by transcriptional (and/or translational) control would also require relatively rapid and eventually regulated enzyme protein degradation. For NR it has been shown that the enzyme has a short half-life of several hours only. It seems reasonable if the control of NR activity and the control of NR synthesis and degradation are coupled.
NR synthesis in spinach leaves was high in the light and low in the dark, and degradation of 35S-methionine-labelled NR was more rapid in the dark than in the light (Weiner and Kaiser, 1999). Further, any treatment activating NR in the dark (anoxia, respiration inhibitors, 5'-AMP-analogues, cellular acidification etc; Kaiser and Huber, 2001) also kept the total NR level high, eventually by preventing degradation. That has led to the suggestion that the 14-3-3–P-NR complex is the preferred substrate for degradation (Kaiser and Huber, 1997). Similarly, in excised tobacco leaves fed with sucrose, the activated NR protein appeared more stable (Morcuende et al., 1998).
NR degradation was also observed in vitro in crude leaf extracts (Weiner and Kaiser, 1999). In extracts from illuminated leaves, NR was relatively stable whereas it was degraded in extracts from darkened leaves. Immunodepletion of 14-3-3 proteins from dark extracts prevented NR degradation, supporting the idea that 14-3-3 binding is an important trigger for degradation.
However, there are also data opposing this view. Cotelle et al. showed that in sugar-starved Arabidopsis cells, NR and other proteins lost their 14-3-3 binding property and were rapidly degraded (Cotelle et al., 2000). At the same time, sugar starvation induced an MG132-sensitive protease. This protease could only cleave target proteins in vitro when they were free of 14-3-3s, but they were perfectly protected when they were phosphorylated and bound to 14-3-3s. The suggestion was that 14-3-3 release may be a signal initiating proteolysis by the sugar starvation-induced protease. Indeed, at least under some conditions that would activate and stabilize NR (see above), total concentrations (per FW) of free sugars in leaves were higher than in normal, darkened leaves where sugar pools are rapidly exhausted (unpublished results, but compare review by Kaiser and Huber, 2001). In none of these experiments was the subcellular location of sugars determined. Thus, up to now it is not clear whether high levels of sugars and/or sugar phosphates in the cytosol, high NR activation state and high NR stability are parallel and independent events or whether they are causally (and sequentially) related. In preliminary experiments (not shown) no inhibition was found of NR degradation in a normal dark phase by MG132, the proteosome blocker that prevented NR degradation in sugar-starved Arabidopsis cells (Cotelle et al., 2000).
As already mentioned, in darkened leaves NR can be activated at least partially by feeding sugars at high concentrations (Table 1). Sugar feeding also kept NR activity (+EDTA) high, indicating either an induction of NR synthesis in the dark or of slowed NR degradation. When sugars were fed together with a PP2A inhibitor, NR was strongly inactivated as in sugar-free controls, yet sugar—and sugar phosphate levels—remained above control levels. Under these conditions, maximum NR activity (which indicates the amount of functional NR protein) decreased almost as rapidly as in the controls (Table 1). That may support this study's original suggestion that in a normal dark phase it is indeed the activation state (or 14-3-3 binding) which controls NR degradation, not the sugar level per se. However, as PP2A inhibitors may also affect NR transcript accumulation and NR synthesis (Redinbaugh et al., 1996), final conclusions appear premature. Also, under prolonged darkness, things may be complicated further by the finding that the abundance of 14-3-3 proteins decreases slowly (Markiewicz et al., 1996). But on the other hand it has been shown previously that, even after several days of continuous darkness when NR had been completely degraded and leaves were virtually NR-free, spinach leaf extracts contained enough protein kinase activity and 14-3-3s to permit an ATP-dependent inactivation of added purified spinach NR that proceded at normal velocity (Glaab and Kaiser, 1996).
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An unusual case: NR from Ricinus communis |
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TopAbstractIntroductionPost-translational NR...Some new insights into...Many external factors trigger...Other ways to inactivate...Are NR modulation and...An unusual case: NR...Potentially important side...References |
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Many NRs from different taxa of higher plants follow the above-described post-translational regulation pattern and, based on the idea that accumulation of toxic products has to be avoided, it was not expected that there would be any exception among higher plants. It was therefore surprising that NR from leaves and roots of Ricinus communis behaved differently from all others. When extracted and measured under standard conditions (pH 7.6, 5–10 mM Mg2+), Ricinus NR was always extremely inactive with only a little difference in light and dark (Kandlbinder et al., 2000
). This high Mg2+ sensitivity of Ricinus NR was maintained after partial purification of Ricinus NR by anion exchange chromatography. By contrast, the maximum activity measured in the presence of EDTA was normal. Such strong inactivation should actually lead to a rapid NR degradation (see above), which was not observed. Measuring NR activity in mixtures of spinach and Ricinus extracts gave no indication for the presence of inhibitory compounds, or of a partial proteolysis of spinach NR in Ricinus extracts (Kandlbinder et al., 2000), nor of a conversion from a low Mg2+-sensitivity into a high sensitivity form (Lillo, 1993). However, unlike with spinach, NR-activity in extracts from Ricinus was strongly increased by pH values below 7, and was extremely low at pH
7.3. This effect was actually due to drastic pH-dependent changes in the Mg-sensitivity of the Ricinus enzyme. At pH 7.6 and pH 6.8, 50% inhibition of NR in spinach leaf extracts occurred at 3 mM and 1 mM Mg2+, respectively. With Ricinus leaf extracts, 50% inhibition was obtained at less than 0.2 mM (pH 7.6), but 5 mM Mg2+ were required at pH 6.8. Preincubation of Ricinus-NR with MgATP gave very little additional inactivation, and removal of 14-3-3s by partial purification of NR, which activates NR from spinach, did not activate Ricinus NR. It thus appears that Ricinus NR is (at pH above 7) always very Mg-sensitive even in the absence of 14-3-3s. Thus, the high Mg-sensitivity of Ricinus NR does not require 14-3-3 binding as in other plant NRs. Whether NR phosphorylation is required is not yet clear. These differences are schematically summarized in Fig. 1
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Immunological studies (Kandlbinder et al., 2000) and DNA sequence analysis (Chyn-Bey Tsai, R Kaldenhoff, WM Kaiser, unpublished results) have indicated that Ricinus-NR has a ‘normal’ serine phosphorylation site and 14-3-3 binding motif. Therefore, the molecular basis and the physiological relevance for the deviating properties of the Ricinus enzyme are not yet understood. In that context it was interesting, however, that Ricinus leaves kept under anoxia in the dark (where the cytosol acidifies) accumulated much higher nitrite concentrations than spinach leaves (Kandlbinder et al., 2000), and they also evolved more nitric oxide (P Rockel, WM Kaiser, unpublished data). That indicates that cytosolic acidification (provoked by anoxia) of Ricinus leaf cells activates NR in vivo relatively more than in spinach, thus supporting the in vitro data.
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Potentially important side reactions of NR: production of NO, reactive oxygen species (ROS) and peroxonitrite |
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TopAbstractIntroductionPost-translational NR...Some new insights into...Many external factors trigger...Other ways to inactivate...Are NR modulation and...An unusual case: NR...Potentially important side...References |
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NR also catalyses the NAD(P)H-dependent reduction of nitrite to nitric oxide (NO). While the basic observation was made more than a decade ago (Klepper, 1987
; Dean and Harper, 1988), the reaction has gained new attention quite recently (Yamasaki et al., 1999; Yamasaki and Sakihama, 2000). One reason for this reborn interest was that knowledge about important functions of NO in animals and also in plants has dramatically increased (for review see Leshem, 2000). Another reason is probably based on the new insights described above on the rapid regulation of NR activity. It was also shown previously that NR can reduce molecular oxygen (Ruoff and Lillo, 1990; Barber and Kay, 1996) to superoxide, and Yamasaki's group showed, in addition, that in the presence of oxygen and NAD(P)H, purified NR also produced peroxonitrite (Yamasaki and Sakihama, 2000) (Fig. 2).
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By following NO emission from enzyme solutions in vitro or from intact leaves or plants, it was shown that the Km for nitrite was 100 µM, and that the reaction was competitively inhibited by nitrate (Ki=50 µM; Rockel et al., 2002
). Accordingly, the rate of NO production was high whenever nitrate reduction exceeded nitrite reduction such that nitrite accumulated. In leaves, nitrite reduction is usually low or absent in the dark. When leaves were exposed to anoxia in the dark, which activates NR, nitrite accumulates even though nitrate reduction rates are far below the potential activity of NR. Under these conditions, NO emission from the leaves was 10–100-fold higher than in air, where NR is more inactivated and nitrite concentrations remain low.
As an example, typical rates of NO-emission from detached spinach leaves (petiole in water) into purified air, were (Rockel et al., 2002): dark: 0.2–0.5 nmol g-1 FW h-1; light: 1–2 nmol g-1 FW h-1; dark, anoxia: 10–50 nmol g-1 FW h-1. NO emission from leaves was also transiently increased for up to 30 min following a light–dark transient (not shown). The shape and duration of the ‘light-off peak’ reflected the transient nitrite accumulation that occurred when the light was abruptly switched off (not shown; Riens and Heldt, 1992).
By feeding nitrite directly through the petiole of detached leaves, high rates of NO emission could be established in the dark. Increasing the NR activation state by any of the above-mentioned means (anoxia, AICAR feeding, sugar feeding) also increased NO emission. On the other hand, inactivating NR in vivo by PP2A inhibitors also decreased the rate of NO emission (P Rockel et al., unpublished results). Thus it seems obvious that (a) whole chain electron transport through NR is required to reduce nitrite to NO and (b) that the above-described modulation of NR activity by external and internal factors also modulates NO-production from nitrite and NADH, both in vitro and in vivo.
As NO production from nitrite by purified NR or by crude leaf extracts was competitively inhibited by nitrate, it is suggested that nitrite and nitrate are reduced on NR at the same site.
Still, even the highest rates of NO emission from nitrite-fed leaves in vivo represented only about 1% of the maximum NR activity. This was confirmed by in vitro measurements of NO production by purified NR, where maximum rates of NO production from NAD(P)h plus saturating nitrite were again only 1–2% of the nitrate reduction capacity of the enzyme (Rockel et al., 2002). Interestingly, these rates are close to maximum rates obtained for NR (Chlorella)-catalysed oxygen reduction to superoxide, which was 0.5% of the nitrate reduction activity (Barber and Kay, 1996). Even these relatively low rates are well in the range of measured NOS activity. In bacterial-infected tobacco leaves, for example, NOS activity in leaf extracts was up to 3 pmol mg-1 protein min-1, which corresponds to about 1.8–3.6 nmol g-1 FW h-1 (Leshem, 2000). It should be noted that these rates are a measured enzyme activity under substrate saturation, whereas the above rates of NO emission from spinach leaves probably underestimate the NO synthesis rate by NR, as part of the NO produced may have reacted already with plant material, and only the excess was emitted. On the other hand, pathogen attacks may cause very localized NO production and thus localized high NO concentrations. Unfortunately, up to now it has not been examined whether and how the NR-system would respond to pathogen attacks. Preliminary experiments where tobacco leaves were treated with the elicitor cryptogein, or with tobacco mosaic virus (tmv), gave no clear effects on NR-dependent NO emission (not shown).
Besides a possible role in plant–pathogen interactions, NO gains increasing attention as signalling molecule eventually involved in controlling growth, differentiation and senescence. It will be important to find out in future research whether and how NO production via NR is affected by abiotic and biotic external factors, and how NR-derived NO interacts with NOS-like activity and ROS formation in plants (Wendehenne et al., 2001; Delledonne et al., 2001).
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Acknowledgments |
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This work was supported by the DFG (SFB 251 and Ka 456/12-1), and EU BIO4 CT 97 2231. Chyn-Bey Tsai is recipient of a DAAD-fellowship. The skilled technical assistance of Eva Wirth and Maria Lesch is gratefully acknowledged.
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Notes |
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4 To whom correspondence should be addressed. Fax: +499318886158. E-mail: kaiser{at}botanik.uni\|[hyphen]\|wuerzburg.de
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Abbreviations |
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NR, nitrate reductase activity; NiR, nitrite reductase activity; NOS, nitric oxide synthase; ROS, reactive oxygen species; PP2A, protein phosphatase 2A.
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References |
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TopAbstractIntroductionPost-translational NR...Some new insights into...Many external factors trigger...Other ways to inactivate...Are NR modulation and...An unusual case: NR...Potentially important side...References |
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