Journal of Experimental Botany, Vol. 53, No. 366, pp. 103-110,
January 1, 2002
© 2002 Oxford University Press
Original Papers |
Regulation of nitric oxide (NO) production by plant nitrate reductase in vivo and in vitro
1 Forschungszentrum Jülich GmbH, Institut für Biologie des Stoffaustauschs, 52425 Jülich, Germany
2 Forschungszentrum Jülich GmbH, Institut für Chemie der Belasteten Atmosphäre, 52425 Jülich, Germany
3 Julius-von-Sachs-Institut für Biowissenschaften, Lehrstuhl für Molekulare Pflanzenphysiologie und Biophysik, Universität Würzburg, Julius-von-Sachs-Platz 2, 97082 Würzburg, Germany
Received 14 June 2001; Accepted 21 August 2001
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
NO (nitric oxide) production from sunflower plants (Helianthus annuus L.), detached spinach leaves (Spinacia oleracea L.), desalted spinach leaf extracts or commercial maize (Zea mays L.) leaf nitrate reductase (NR, EC 1.6.6.1) was continuously followed as NO emission into the gas phase by chemiluminescence detection, and its response to post-translational NR modulation was examined in vitro and in vivo. NR (purified or in crude extracts) in vitro produced NO at saturating NADH and nitrite concentrations at about 1% of its nitrate reduction capacity. The Km for nitrite was relatively high (100 µM) compared to nitrite concentrations in illuminated leaves (10 µM). NO production was competitively inhibited by physiological nitrate concentrations (Ki=50 µM). Importantly, inactivation of NR in crude extracts by protein phosphorylation with MgATP in the presence of a protein phosphatase inhibitor also inhibited NO production. Nitrate-fertilized plants or leaves emitted NO into purified air. The NO emission was lower in the dark than in the light, but was generally only a small fraction of the total NR activity in the tissue (about 0.01–0.1%). In order to check for a modulation of NO production in vivo, NR was artificially activated by treatments such as anoxia, feeding uncouplers or AICAR (a cell permeant 5'-AMP analogue). Under all these conditions, leaves were accumulating nitrite to concentrations exceeding those in normal illuminated leaves up to 100-fold, and NO production was drastically increased especially in the dark. NO production by leaf extracts or intact leaves was unaffected by nitric oxide synthase inhibitors. It is concluded that in non-elicited leaves NO is produced in variable quantities by NR depending on the total NR activity, the NR activation state and the cytosolic nitrite and nitrate concentration.
Key words: Helianthus annuus, nitrate reductase, nitric oxide, protein phosphorylation, Spinacia oleracea.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Nitric oxide (NO) is an inorganic free radical that acts as a signalling molecule with multiple biological functions in vertebrates. There it is produced mainly by nitric oxide synthase (NOS), which catalyses NO and L-citrulline formation from O2 and L-arginine. The NOS activity was also detected in higher plant extracts (Cueto et al., 1996
; Ninnemann and Maier, 1996; Ribeiro et al., 1999) and peroxisomes (Barroso et al., 1999). NO production by plant tissues was first observed by Klepper (Klepper, 1975a) with soybean plants treated with photosynthetic inhibitor herbicides (Klepper, 1978, 1979) or other chemicals (Klepper, 1990, 1991) as well as under dark anaerobic conditions (Klepper, 1987, 1990). It was suggested that this emission was due to chemical reactions of accumulated nitrite with plant metabolites such as salicylate derivatives or the chemical decomposition of HNO2.
NO was also observed to be the predominant compound evolved during a purged in vivo assay with soybean nitrate reductase derived from accumulated nitrite (Harper, 1981). Results obtained with boiled leaflets (Harper, 1981) and the mutant soybean line nr1 (Dean and Harper, 1986, 1988; Nelson et al., 1983; Ryan et al., 1983) further indicated that the enzymatic reaction of a constitutive NAD(P)H:nitrate reductase is responsible for the evolution of NOx. Experiments with 15N-labelled nitrate as the substrate for nitrate reduction showed that NOx is produced from 15
(Dean and Harper, 1986). More recently, it was shown that NR from maize could also produce NO from nitrite plus NADH, and that this reaction could be prevented by azide (Yamasaki et al., 1999; Yamasaki and Sakihama, 2000). These authors also found NO production with nitrate as the substrate, but in that case with a time-lag. They concluded that the actual substrate for NR-dependent NO production was nitrite, not nitrate.
It is known that different nitrate-nourished plant species have a compensation point for NO uptake from the atmosphere showing a net NO emission at low atmospheric NO concentrations (Rockel et al., 1996; Wildt et al., 1997).
Recent studies suggested that nitric oxide may play a role as a messenger in plant pathogen resistance (Pfeiffer et al., 1994; Delledonne et al., 1998; Durner and Klessig, 1999; Bolwell, 1999) by increasing cGMP and salicylic acid levels. It can further influence plant growth (Leshem and Haramaty, 1996; Beligni and Lamattina, 2000; Leshem, 2000), protect against cytotoxicity of reactive oxygen species (Beligni and Lamattina, 1999) and cause the accumulation of phytoalexin (Noritake et al., 1996).
In spite of increasing evidence for important physiological roles of NO in plants, it is still unclear whether and under which conditions NO is produced by nitrate reductase (or by NOS), and how this is regulated. Here, evidence is provided that post-translational modulation of NR in vitro and in vivo also modulates the NO production rates. It is also shown (i) that NR-dependent NO production under some conditions may drastically exceed the published activities of NOS in leaves, (ii) that it has a relatively high Km for nitrite of 100 µM, and (iii) that it is competitively inhibited by low nitrate concentrations (Ki=50 µM) or by chlorate, both in vitro and in vivo.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Plant material and growth
Sunflower (Helianthus annuus L. var. giganteus) and spinach (Spinacia oleracea L. var. Monnopa) were germinated in moist quartz sand at a 14 h photoperiod (280 µmol m-2 s-1) with a temperature regime of 28/23 °C (day/night) and 90/80% relative humidity. After 1–2 weeks the plants were transferred to a hydroponic system (Rockel, 1997
) and grown at 20 °C and 60% relative humidity during a 12 h photoperiod (350 µmol m-2 s-1) and 17 °C and 80% relative humidity during darkness, respectively. The aerated nutrient solution was automatically adjusted to pH 6.0 by the addition of 0.1 N NaOH (
nutrient solution) or 0.1 N H2SO4 (
nutrient solution). Nutrient concentrations were kept constant (±5%) by the automated addition of a 10-fold stock solution (Rockel, 1997). The nutrient solution for nitrate-grown plants contained 0.2 mM KNO3, 0.25 mM Ca(NO3)2, 0.05 mM NaNO3, 0.05 mM KH2PO4, and 0.1 mM MgSO4; the nutrient solution contained 0.05 mM (NH4)2HPO4, 0.35 mM (NH4)2SO4, 0.125 mM K2SO4, 0.1 mM MgSO4, and 0.25 mM CaCl2; the N-free nutrient solution contained 0.05 mM KH2PO4, 0.125 mM K2SO4, 0.1 mM MgSO4, and 0.25 mM CaCl2. Micronutrient concentrations in all solutions were 10 µM KCl, 6.3 µM H3BO3, 4.0 µM FeH2-EDTA, 0.4 µM ZnSO4.7H2O, 0.4 µM MnSO4.H2O, 0.1 µM Na2MoO4.2H2O, and 0.1 µM CuSO4.5H2O.
Experimental set-up for gas phase NO measurements
Whole plants were transferred to the experimental set-up for NO measurements when they had reached a total leaf area of approximately 500–1000 cm2.
The upper part of the plant was then placed into a continuously stirred tank reactor, a glass vessel of 164 l volume through which purified air was passed with a constant flow of 40 l min-1. A teflon plate sealed the glass vessel from the environment and held the plant in position. Background concentrations of ozone, NO and NO2 of the inflowing air were less than 100 ppt. Concentrations of NO and NO2 in the air were measured by chemiluminescence detection (CLD AL ppt 770, Eco-physics, Dürnten, Switzerland, detection limit 20 ppt; 1 min time resolution). Water vapour and CO2 were detected by an infrared analyser (Rosemount, Hanau, Germany, Binos 100). The ozone concentration was measured by UV absorption (Dasibi, Glendale, CA, USA, 1008 RS). All gas concentrations of the inflowing and outflowing air were measured alternately at 1 h intervals as described previously (Neubert et al., 1993). NO emission fluxes were calculated from the concentration differences between the inlet and outlet of the chamber (Wildt et al., 1997). All experiments were carried out with nitrate-grown plants unless otherwise stated.
Single leaf experiments
For single leaf experiments, the leaves were cut off from the plant and immediately placed in nutrient solution, where the petiole was cut off a second time below the solution surface. The leaves were then placed in a small glass chamber (vol. 5.0 l) with an airflow of 3–5 l min-1. The CO2 concentration and water vapour were continuously measured between the inlet and outlet. The NO concentration of the inflowing air was measured before and after each experiment, whereas the outflowing air was measured continuously during the experiments.
Preparation of leaf extracts
Leaves without midrib were weighed and immediately ground to a fine powder in liquid nitrogen. For desalted extracts, an extraction buffer containing 50 mM HEPES-KOH (pH 7.6), 1 mM DTT, 10 µM FAD, and 15 mM MgCl2, was added to the frozen powder (2 ml g-1 FW). Where indicated in the legends, protein phosphatase inhibitors were added in order to maintain the actual state of NR phosphorylation. After centrifugation (16000 g, 10 min, 4 °C), the supernatant was desalted on Sephadex G25 (Sigma, Steinheim, Germany) spin columns (9 ml gel volume, 3.5 ml extract, 4 °C) equilibrated with the extraction buffer. Two ml each of this extract were preincubated at room temperature as described and used for the NO measurement.
NO measurement over solutions
For the measurement of NO production from crude extracts or purified NR, the solutions were placed in a Petri dish (diameter 5 cm) located in a sealed glass chamber (1.0 l volume). All experiments were performed in darkness at 25 °C under aerobic conditions. The sample was continuously stirred, and purified air was passed through the glass chamber with a constant flow of 3.0 l min-1. NO was measured at the inlet and outlet of the chamber by chemiluminescence detection as described above. Effectors were added as indicated without disturbing the assay conditions. All compounds were also tested without NR or extract to exclude possible chemical NO formation.
Nitrate reductase activity and nitrite content
For separate determinations of NR activity, leaves without midrib were weighed and immediately ground in liquid nitrogen. Two ml of extraction buffer containing 50 mM HEPES-KOH (pH 7.6), 1 mM DTT, 10 µM FAD, and 15 mM MgCl2, were added to 1 g of frozen powder. After centrifugation (16000 g, 10 min, 4 °C), aliquots of the extract were directly used for the colorimetric determination of the original nitrite content of the leaves. The remaining supernatant was desalted on Sephadex G25 spin columns (1.5 ml gel volume, 0.650 ml extract, 4 °C) equilibrated with the extraction buffer. Aliquots (100 µl) were added to a reaction medium (900 µl, 50 mM HEPES-KOH (pH 7.6), 10 µM FAD, 1 mM DTT, 15 mM MgCl2, 5 mM KNO3, 0.2 mM NADH) and incubated for 3 min at 25 °C. After 3 min, the reaction was stopped by adding zinc acetate (125 µl, 0.5 M). Following centrifugation (16000 g, 10 min), the supernatant was treated with 10 µM phenazine methosulphate (20 min, dark) to oxidize unreacted NADH, and used for colorimetric determination of nitrite formed as described earlier (Hageman and Reed, 1980).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
In vitro NO production by NR can be measured as NO emission from solution into the gas phase
In order to test whether the emission of NO into the gas phase from enzyme solutions could be used as a continuous measurement for the NO production, previous work carried out with a NO-analyser (Yamasaki and Sakihama 2000
), using purified, commercially available maize leaf NAD(P)H-NR (Sigma, Deisenhofen, Germany. Lot Nr N 2397) was first repeated. The measurement of nitrite reductase activity in these preparations were negative, and addition of L-NNA to the assay indicated no contamination with NOS (data not shown). 10 mU maize leaf NR (according to the manufacturers specification) was added to 5 ml of a reaction medium containing nitrate (0.1 mM) plus NADH (0.1 mM), and the NO production was monitored continuously. Every 10 min, 100 µl aliquot was removed from the mixture for nitrite determination.
The results confirm recent data (Yamasaki and Sakihama, 2000) which were obtained by direct measurements of NO in solution (either with an NO analyser or by fluorescence indicators), and are only briefly summarized below. With nitrate as a substrate, NO emission began after a lag phase of 30 min when a nitrite concentration of between 50 and 60 µM had been reached. As already concluded (Yamasaki and Sakihama, 2000), NO was obviously not directly formed from nitrate, but from nitrite. In these experiments, where the maximum initial nitrite production rate (or NR activity) was 10 nmol min-1, the maximum NO production rate (reached towards the end of the experiment) was only (0.09 nmol min-1). Thus, the NO production by commercial purified maize NR was slightly less than 1% of the NR activity of the preparation. Similarly, the kinetics of NO emission were examined after the addition of nitrite, in the complete absence of nitrate (data not shown). The addition of 100 µM nitrite to 5 ml buffer solution containing NR and NADH as before resulted in an immediate onset of NO emission from the solution with similar rates as before, again confirming previous measurements (Yamasaki and Sakihama, 2000).
The substrate (nitrite) affinity of that commercial maize leaf NR for NO production was also examined (Fig. 1). The Km for nitrite was approximately 100 µM. Consequently, it was found that NO production was competitively inhibited by low nitrate concentrations with a Ki=50 µM. Chlorate as a well-known competitive (to nitrate) inhibitor of NR also caused a strong inhibition of NO production from nitrite (data not shown). In contrast, other anions, for example, 100 µM sulphate, had no effect on NO production.
|
) or presence of 50 µM nitrate (
).
Modulation of NO production by modulation of NR activity in vitro
As already mentioned, an aim of this study was to discover whether the well-known modulation of NR activity by reversible protein phosphorylation and 14-3-3-binding would also affect NO production with nitrite and NADH as substrates. It has previously been shown that the NR activity (measured in the presence of Mg2+) can be rapidly modulated in vitro by preincubation of leaf extracts with MgATP or various effectors (for review see Kaiser et al., 1999). In order to examine whether NO production would also be modulated by ATP preincubation, desalted leaf extracts were used instead of purified NR, as auxiliary enzymes (NR kinase, P-NR phosphatase and 14-3-3 proteins) are required for the modulation. For the experiment depicted in Fig. 2, a freshly prepared desalted extract from illuminated spinach leaves was preincubated with 5'-AMP plus EDTA, which causes the full activation of NR by dephosphorylation and release of 14-3-3-proteins (Kaiser et al., 1999). Following the addition of NADH plus nitrite in 2 ml of a stirred reaction medium, NO was emitted into the gas phase above the solution.
|
Approximately 10 min after substrate addition, the NO emission decreased again. In contrast, preincubation of the extract with ATP, Mg2+ and protein phosphatase 2A inhibitor cantharidine, which inactivates NR by converting it into the catalytically inactive P-NR-14-3-3 complex (Kaiser et al., 1999
), caused an almost complete inhibition of nitrite-dependent NO production. In the above example, the measured NRA in an aliquot of the extract was 10.3 µmol nitrite g-1 FW h-1 for the AMP pretreated samples and 1.4 µmol nitrite g-1 FW h-1 after preincubation with ATP plus cantharidine. The rates of NO emission from nitrite of the fully activated enzyme on a fresh weight basis were 4.3 nmol NO g-1 FW h-1, and thus again only a small fraction of NR activity in the crude extract, as observed with the commercial purified maize NR.
Regulation of NO emission in vivo
Sunflowers adapted to constant nitrate nutrition usually emitted no significant amounts of NO in the dark. In the light period up to 2 nmol NO g-1 FW h-1 were released. Figure 3 shows a typical dark–light cycle of intact nitrate-grown sunflower plants.
|
When detached sunflower leaves with the petioles in nitrate solution (10 mM) were illuminated in air, NO was emitted at a constantly low rate (Fig. 4
). When the light was switched off, NO release transiently increased, but again settled at a very low steady-state value in the dark. This ‘light-off burst’ of NO emission was routinely observed not only with sunflower, but also with leaves or intact plants from other species like spinach or tobacco (data not shown). It was usually correlated with a transient nitrite accumulation (Fig. 4), (also compare Riens and Heldt, 1992).
|
–) (nmol NO g-1 FW h-1) and nitrite content (•) of a detached sunflower leaf in the exposure-chamber. At t=0 light was switched off.The above in vitro experiments with desalted leaf extracts have shown that NO production by NR from nitrite follows the modulation of NR by protein phosphorylation/dephosphorylation. NR can also be modulated in vivo. The activity is usually high under good photosynthetic conditions and is decreased in the dark (Fig. 5
). In the dark, NR can be activated to or above the light level by various treatments such as anoxia or by feeding uncouplers or cell permeable 5'-AMP analogues (Kaiser et al., 1999). All these treatments also cause nitrite accumulation in darkened leaves or in roots (Kaiser et al., 1993; Botrel and Kaiser 1997), and should therefore be expected to increase NO emission. This was indeed observed. One example is shown in Fig. 5, which gives NR activity (+Mg2+), nitrite contents and NO emission by intact leaves.
|
As before, NRA in the dark was only about 50% of that in illuminated leaves, and the darkened leaves also contained 50% less nitrite than in the light. This was paralleled by NO emission. Under anoxia in the dark, NRA was strongly activated reaching approximately four times the activity of that in air. However, the leaf nitrite content increased more than 100-fold within 3 h, as did the NO emission (Fig. 5
). As seen in vitro, even the maximum rate of NO release was only a small fraction (
1%) of the NR activity in the extract, although nitrite concentrations in the tissue (up to 4.8 µmol g-1 FW, corresponding to a concentration in the water phase above 5 mM) were far above the Km nitrite (100 µM). Thus, NO release in vivo basically followed the pattern of NR activity, and was also related to the nitrite concentration in the tissue.
Table 1 summarizes the influence of various other methods of activating and inactivating NR in the dark. Dinitrophenol (DNP) is an uncoupler of oxidative phosphorylation and activates NR. Such activation of NR by uncouplers in the dark (air) has been shown to result in nitrite accumulation, similar to anoxia (Kaiser et al., 1993). In vitro, inactive (phosphorylated) NR can be activated by 5'-AMP (Kaiser et al., 1999). In vivo, this effect can be mimicked by feeding the cell permeable 5-aminoimidazole-4-carboxyamide 1-ß-D-ribofuranoside (AICAR), which is converted inside the cell to the phosphorylated AMP-analogue ZMP (5-aminoimidazole-4-carboxyamide-1-ß-D-ribofuranosyl 5'-monophosphate). Leaves fed with up to 20 mM AICAR in the dark show a very high activation state of NR (Huber and Kaiser, 1996). Further, the activation state of NR can be decreased by preventing dephosphorylation with okadaic acid, a specific inhibitor of PP2A phosphatases (Kaiser et al., 1999). As expected, okadaic acid decreased the light-dependent NO emission.
|
The authors were also interested to examine whether nitric oxide synthase (NOS) would contribute to the NO emission from leaves. As the NOS reaction depends on the presence of oxygen, anoxia could not be used for inducing NO emission. Instead, the effect of the NOS inhibitor N-
-nitro-L-arginine (L-NNA) was examined on the high NO emission in response to DNP. L-NNA had no inhibitory effect on the NO emission after DNP treatment. In summary, all treatments which activated NR in the dark and also caused a nitrite accumulation to various degrees, led to increased NO emission, while okadaic acid completely prevented NO emission.
As nitrate was a very effective inhibitor of NO production in vitro, it was predicted that an increase in the cytosolic nitrate concentration would also decrease the rate of NO production in intact leaves. This was indeed confirmed, at least qualitatively. When a high rate of NO emission from intact leaves was initiated by anoxia in the dark, feeding 50 mM KNO3 through the leaf petiole caused an almost immediate decrease in NO emission (Fig. 6), which recovered slowly after a certain period of time. Here, cytosolic nitrate concentrations were unknown and therefore, the size and kinetics of the inhibition could not be predicted.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
These measurements confirm previous results that NO can be produced by NR from nitrite and NADH. To what extent different isoforms of NR are involved has not yet been examined, but in the leaves tested, NADPH-dependent activity was negligible (not shown).
NO may also be formed non-enzymatically. Mallick et al. assumed that NO formed from illuminated algae suspensions was at least partly due to a reaction of nitrite with reductants such as ascorbate or glutathione, as shown by NO production in pure nitrite solutions with these reductants (Mallick et al., 2000). On the other hand, NO has been shown to react with glutathione to form N-nitrosoglutathione, which may even represent a storage form for NO (Singh et al., 1999). Further, algal cells release nitrite to the medium, where it may be also photolysed to NO under high light intensities. Indeed, when leaves were fed with high nitrite concentrations in strong light, some NO emissions were found which did not respond to NR-inactivation (data not shown). However, in normal, nitrate-fed leaves NO production could be completely prevented by NR inactivation by feeding high nitrate or chlorate concentrations (see above). Thus, in the experiments depicted above, NO was exclusively formed by a reaction catalysed by NR.
Generally, even at saturating substrate concentrations, NO production appeared to be only a small fraction (1%) of the nitrate-reducing capacity of NR. Nitrate was not a direct substrate, but a highly efficient competitive inhibitor. The apparent Km for nitrite (100 µM) was above the normal nitrite concentration found in illuminated leaves, which are approximately 10 nmol g-1 FW, but cytosolic nitrite concentrations are unknown. Nitrate concentrations in the cytosol of root cells are usually in the millimolar range (Miller and Smith, 1996), and similar concentrations have been found in leaf mesophyll cells (AJ Miller, personal communication). Since nitrite-dependent NO production is competitively inhibited by nitrate, NO emission rates from intact leaves must be very low, as observed (0.01–0.1% of the NR activity). In certain situations, however, for example, when NR was activated to such an extent that nitrite production exceeded the rate of nitrite reduction, the maximum NO emission rates from leaves came close to the maximum values (under substrate saturation) obtained with purified NR or leaf extracts in vitro.
reduction to NO by NR is probably a one-electron-transfer reaction. So far, nothing is known about its molecular details. It is also unknown, why this reaction appears to be limited to such a low fraction of the nitrate reduction capacity of the enzyme. Due to that low capacity of NR for NO production it seems improbable that the reaction is suited to avoid excessive nitrite accumulation. Nevertheless, the system has an extremely broad response range, making it suitable, theoretically, for contributing to NO signalling. NO production by NOS, assayed with L-[3H]-citrulline as substrate, was found to be 3 pmol mg-1 leaf protein min-1, for example, in bacterial-infected tobacco leaves (Leshem, 2000). This is equivalent to about 1.8 nmol g-1 FW h-1 (a mean tobacco leaf protein concentration is 10–20 mg g-1 FW, not shown). In these experiments, NO production rates by NR in vivo were between 2–200 nmol g-1 FW h-1. Thus, the capacity of the NR-system in vivo appears well in the range of NOS-activity, and may exceed it up to 100-fold under some conditions. Certainly participation of NR-derived NO in cellular signalling or interference with it would require strict regulation and sensitivity to effectors. It was therefore important to show here for the first time that the post-translational modulation of NR by serine phosphorylation and binding of a 14-3-3 dimer (for review see Kaiser and Huber, 1997) did indeed modulate NO production by NR, in vivo and in vitro. In vivo, the activation of NR would increase nitrite concentrations to the extent that nitrite reduction cannot cope, and this would increase NO production, because normal nitrite concentrations are far below the Km. However, activation of NR also increased NO production from added (saturating) nitrite in vitro. Thus, the modulation of NR has a 2-fold effect on NO production: a direct one by modulating the electron flow through the NR enzyme, and an indirect one by affecting the nitrite concentration. In addition, conditions changing the cytosolic nitrate concentration (which is a competitive inhibitor) or the rate of nitrite reduction must have drawbacks on the NR-dependent NO production. Thus, the system has multiple sites of interaction with photosynthesis and metabolism. Future work will focus on both the molecular mechanism and the regulation of NO production, on its physiological consequences. and its relation to the second NO-producing system, NOS.
|
Acknowledgments |
|---|
This work was supported by the DFG (KA 456/12-1, WI 982/2-1).
|
Notes |
|---|
4 To whom correspondence should be addressed. Fax: +49 2461 612492. E-mail: P.Rockel{at}fz\|[hyphen]\|juelich.de
|
Abbreviations |
|---|
NR, nitrate reductase; NRA, NR-activity; P-NR, phosphorylated NR; NO, nitric oxide; NOS, NO-synthase; AICAR, 5-aminoimidazole-4-carboxyamide; DNP, dinitrophenole; ZMP, 5-aminoimidazole-4-carboxyamide-1-ß-D-ribofuranosyl 5'-monophosphate; L-NNA, N-
–nitro-L-arginine.. |
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Barroso JB, Corüas FJ, Carreras A, Sandalio LM, Valderrama R, Palma JM, Lupianez JA, del Rio LA. 1999. Localization of nitric oxide synthase in plant peroxisomes. The Journal of Biological Chemistry 274, 36729–36733.
Beligni MV, Lamattina L. 1999. Nitric oxide counteracts cytotoxic processes mediated by reactive oxygen species in plant tissues. Planta 208,337–344.[Web of Science]
Beligni MV, Lamattina L. 2000. Nitric oxide stimulates seed germination and de-etiolation and inhibits hypocotyl elongation, three light inducible responses in plants. Planta 210, 215–221.[Web of Science][Medline]
Bolwell GP. 1999. Role of active oxygen species and NO in plant defence responses. Current Opinion in Plant Biology 2, 287–294.[Web of Science][Medline]
Botrel A, Kaiser WM. 1997. Nitrate reductase activation state in barley roots in relation to the energy and carbohydrate status. Planta 201, 496–501.[Web of Science][Medline]
Cueto M, Hernandez-Perera O, Martin R, Bentura ML, Rodrigo J, Lamas S, Golvano MP. 1996. Presence of nitric oxide synthase activity in roots and nodules of Lupinus albus. FEBS Letters 398, 159–164.[Web of Science][Medline]
Dean JV, Harper JE. 1986. Nitric oxide and nitrous oxide production by soybean and winged bean during in vivo nitrate reductase assay. Plant Physiology 82, 718–723.
Dean JV, Harper JE. 1988. The conversion of nitrite to nitrogen oxide(s) by the constitutive NAD(P)H-nitrate reductase enzyme from soybean. Plant Physiology 88, 389–395.
Delledonne M, Xia Y, Dixon RA, Lamb C. 1998. Nitric oxide functions as a signal in plant disease resistance. Nature 394, 585–588.[Medline]
Durner J, Klessig DF. 1999. Nitric oxide as a signal in plants. Current Opinion in Plant Biology 2, 369–374.[Web of Science][Medline]
Hageman RH, Reed AJ. 1980. Nitrate reductase from higher plants. Methods in Enzymology 69C, 270–280.
Harper JE. 1981. Evolution of nitrogen oxide(s) during in vivo nitrate reductase assay of soybean leaves. Plant Physiology 68, 1488–1493.
Huber SC, Kaiser WM. 1996. 5-Aminoimidazole-4-carboxiamide riboside activates nitrate reductase in darkened spinach and pea leaves. Physiologia Plantarum 98, 833–837.
Kaiser WM, Dittrich A, Heber U. 1993. Sulfate concentration in Norway spruce needles in relation to atmospheric SO2: a comparison of trees from various forests in Germany with trees fumigated with SO2 in growth chambers. Tree Physiology 12, 1–13.[Abstract]
Kaiser WM, Huber SC. 1997. Correlation between apparent activation state of nitrate reductase (NR), NR hysteresis and degradation of NR protein. Journal of Experimental Botany 48, 1367–1374.
Kaiser WM, Weiner H, Huber SC. 1999. Nitrate reductase in higher plants: a case study for transduction of environmental stimuli into control of catalytic activity. Physiologia Plantarum 105, 385–390.
Klepper LA. 1975a. Evolution of nitrogen oxide gases from herbicide treated plant tissues. WSSA Abstracts 184, 70.
Klepper LA. 1978. Nitric oxide (NO) evolution from herbicide-treated soybean plants. Plant Physiology 61, Supplement, 65.
Klepper LA. 1979. Nitric oxide (NO) and nitrogen dioxide emissions from herbicide-treated soybean plants. Atmospheric Environment 13, 537–542.
Klepper LA. 1987. Nitric oxide emissions from soybean leaves during in vivo nitrate reductase assays. Plant Physiology 85, 96–99.
Klepper LA. 1990. Comparison between NOx evolution mechanisms of wild-type and nr1 mutant soybean leaves. Plant Physiology 93, 26–32.
Klepper LA. 1991. NOx evolution by soybean leaves treated with salicylic acid and selected derivatives. Pesticide Biochemistry and Physiology 39, 43–48.
Leshem YY. 2000. Nitric oxide in plants. Occurence, function and use. Dordrecht, Boston, London: Kluwer Academic Publishers.
Leshem YY, Haramaty E. 1996. The characterization and contrasting effects of the nitric oxide free radical in vegetative stress and senescence of Pisum sativum L. foliage. Journal of Plant Physiology 148, 258–263.[Web of Science]
Mallick M, Mohn FH, Soeder CJ. 2000. Evidence supporting nitrite-dependent NO release by the green microalga Scenedesmus obliquus. Journal of Plant Physiology 157, 40–46.[Web of Science]
Miller AJ, Smith SJ. 1996. Nitrate transport and compartmentation in cereal root cells. Journal of Experimental Botany 47, 843–854.
Nelson RS, Ryan SA, Harper JE. 1983. Soybean mutants lacking a constitutive nitrate reductase activity. I. Selection and initial plant characterization. Plant Physiology 72, 503–509.
Neubert A, Kley D, Wildt J, Segschneider H-J, Förstel H. 1993. Uptake of NO, NO2 and O3 by sunflower (Helianthus annuus L.) and tobacco plants (Nicotiana tabacum L.): dependence on stomatal conductivity. Atmospheric Environment 27A, 2137–2145.
Ninnemann H, Maier J. 1996. Indications for the occurrence of nitric oxide synthases in fungi and plants and the involvement in photoconidiation of Neurospora crassa. Photochemistry and Photobiology 64(2), 393–398.[Web of Science][Medline]
Noritake T, Kawakita K, Doke N. 1996. Nitric oxide induces phytoalexin accumulation in potato tuber tissues. Plant and Cell Physiology 37, 113–116.
Pfeiffer S, Janistyn B, Jessner G, Pichorner H, Ebermann R. 1994. Gaseous nitric oxide stimulates guanosine-3',5'-cyclic monophosphate (cGMP) formation in spruce needles. Phytochemistry 36, 259–262.[Web of Science]
Ribeiro Jr EA, Cunha FQ, Tamashiro WMSC, Martins IS. 1999. Growth phase-dependent subcellular localization of nitric oxide synthase in maize cells. FEBS Letters 445, 283–286.[Web of Science][Medline]
Riens B, Heldt HW. 1992. Decrease of nitrate reductase activity in spinach leaves during a light-dark transition. Plant Physiology 98, 573–577.
Rockel P. 1997. Growth and nitrate consumption of sunflowers in the rhizostat, a device for continuous nutrient supply to plants. Journal of Plant Nutrition 20, 1431–1447.
Rockel P, Rockel A, Wildt J, Segschneider H-J. 1996. Nitric oxide (NO) emission by higher plants. In: Van Cleemput O et al., eds. Progress in nitrogen cycling studies. The Netherlands: Kluwer Academic Publishers, 603–606.
Ryan SA, Nelson RS, Harper JE. 1983. Soybean mutants lacking constitutive nitrate reductase activity. II. Nitrogen assimilation, chlorate resistance and inheritance. Plant Physiology 72, 510–514.
Singh RJ, Hogg N, Goss SPA, Antholine WE, Kalyanaraman B. 1999. Mechanism of superoxide dismutase/H2O2 mediated nitric oxide release from S-nitrosoglutathione role of glutamate. Archives of Biochemistry and Biophysics 372, 8–15.[Web of Science][Medline]
Wildt J, Kley D, Rockel A, Rockel P, Segschneider H-J. 1997. Emission of NO from several higher plant species. Journal of Geophysical Research 102, 5919–5927.[Web of Science]
Yamasaki H, Sakihama Y. 2000. Simultaneous production of nitric oxide and peroxynitrite by plant nitrate reductase: in vitro evidence for the NR-dependent formation of active nitrogen species. FEBS Letters 468, 89–92.[Web of Science][Medline]
Yamasaki H, Sakihama Y, Takahashi S. 1999. An alternative pathway for nitric oxide production in plants: new features of an old enzyme. Trends in Plant Science 4, 128–129.[Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M.-G. Zhao, L. Chen, L.-L. Zhang, and W.-H. Zhang Nitric Reductase-Dependent Nitric Oxide Production Is Involved in Cold Acclimation and Freezing Tolerance in Arabidopsis Plant Physiology, October 1, 2009; 151(2): 755 - 767. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Rodriguez-Serrano, M. C. Romero-Puertas, D. M. Pazmino, P. S. Testillano, M. C. Risueno, L. A. del Rio, and L. M. Sandalio Cellular Response of Pea Plants to Cadmium Toxicity: Cross Talk between Reactive Oxygen Species, Nitric Oxide, and Calcium Plant Physiology, May 1, 2009; 150(1): 229 - 243. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. U. Igamberdiev and R. D. Hill Plant mitochondrial function during anaerobiosis Ann. Bot., January 1, 2009; 103(2): 259 - 268. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Moreau, G. I. Lee, Y. Wang, B. R. Crane, and D. F. Klessig AtNOS/AtNOA1 Is a Functional Arabidopsis thaliana cGTPase and Not a Nitric-oxide Synthase J. Biol. Chem., November 21, 2008; 283(47): 32957 - 32967. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. J. Corpas, M. Chaki, A. Fernandez-Ocana, R. Valderrama, J. M. Palma, A. Carreras, J. C. Begara-Morales, M. Airaki, L. A del Rio, and J. B. Barroso Metabolism of Reactive Nitrogen Species in Pea Plants Under Abiotic Stress Conditions Plant Cell Physiol., November 1, 2008; 49(11): 1711 - 1722. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Seligman, E. E. Saviani, H. C. Oliveira, C. A. F. Pinto-Maglio, and I. Salgado Floral Transition and Nitric Oxide Emission During Flower Development in Arabidopsis thaliana is Affected in Nitrate Reductase-Deficient Plants Plant Cell Physiol., July 1, 2008; 49(7): 1112 - 1121. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. J. Gilberthorpe and R. K. Poole Nitric Oxide Homeostasis in Salmonella typhimurium: ROLES OF RESPIRATORY NITRATE REDUCTASE AND FLAVOHEMOGLOBIN J. Biol. Chem., April 25, 2008; 283(17): 11146 - 11154. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. N. Bouchard and H. Yamasaki Heat Stress Stimulates Nitric Oxide Production in Symbiodinium microadriaticum: A Possible Linkage between Nitric Oxide and the Coral Bleaching Phenomenon Plant Cell Physiol., April 1, 2008; 49(4): 641 - 652. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Besson-Bard, C. Courtois, A. Gauthier, J. Dahan, G. Dobrowolska, S. Jeandroz, A. Pugin, and D. Wendehenne Nitric Oxide in Plants: Production and Cross-talk with Ca2+ Signaling Mol Plant, March 1, 2008; 1(2): 218 - 228. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Vitecek, V. Reinohl, and R. L. Jones Measuring NO Production by Plant Tissues and Suspension Cultured Cells Mol Plant, March 1, 2008; 1(2): 270 - 284. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Neill, J. Bright, R. Desikan, J. Hancock, J. Harrison, and I. Wilson Nitric oxide evolution and perception J. Exp. Bot., January 1, 2008; 59(1): 25 - 35. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Wang, X. Xing, and N. Crawford Nitrite Acts as a Transcriptome Signal at Micromolar Concentrations in Arabidopsis Roots Plant Physiology, December 1, 2007; 145(4): 1735 - 1745. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Sugiura, M. N. Georgescu, and M. Takahashi A Nitrite Transporter Associated with Nitrite Uptake by Higher Plant Chloroplasts Plant Cell Physiol., July 1, 2007; 48(7): 1022 - 1035. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Liu, R. Wu, Q. Wan, G. Xie, and Y. Bi Glucose-6-Phosphate Dehydrogenase Plays a Pivotal Role in Nitric Oxide-Involved Defense Against Oxidative Stress Under Salt Stress in Red Kidney Bean Roots Plant Cell Physiol., March 1, 2007; 48(3): 511 - 522. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Rusterucci, M. C. Espunya, M. Diaz, M. Chabannes, and M. C. Martinez S-Nitrosoglutathione Reductase Affords Protection against Pathogens in Arabidopsis, Both Locally and Systemically Plant Physiology, March 1, 2007; 143(3): 1282 - 1292. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I.G.L. Libourel, P.M. van Bodegom, M.D. Fricker, and R.G. Ratcliffe Nitrite Reduces Cytoplasmic Acidosis under Anoxia Plant Physiology, December 1, 2006; 142(4): 1710 - 1717. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Jasid, M. Simontacchi, C. G. Bartoli, and S. Puntarulo Chloroplasts as a Nitric Oxide Cellular Source. Effect of Reactive Nitrogen Species on Chloroplastic Lipids and Proteins Plant Physiology, November 1, 2006; 142(3): 1246 - 1255. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Planchet and W. M. Kaiser Nitric oxide (NO) detection by DAF fluorescence and chemiluminescence: a comparison using abiotic and biotic NO sources J. Exp. Bot., September 1, 2006; 57(12): 3043 - 3055. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Yamamoto-Katou, S. Katou, H. Yoshioka, N. Doke, and K. Kawakita Nitrate Reductase is Responsible for Elicitin-induced Nitric Oxide Production in Nicotiana benthamiana Plant Cell Physiol., June 1, 2006; 47(6): 726 - 735. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. B Barroso, F. J Corpas, A. Carreras, M. Rodriguez-Serrano, F. J Esteban, A. Fernandez-Ocana, M. Chaki, M. C Romero-Puertas, R. Valderrama, L. M Sandalio, et al. Localization of S-nitrosoglutathione and expression of S-nitrosoglutathione reductase in pea plants under cadmium stress J. Exp. Bot., May 1, 2006; 57(8): 1785 - 1793. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. N. Tun, C. Santa-Catarina, T. Begum, V. Silveira, W. Handro, E. I. S. Floh, and G. F. E. Scherer Polyamines Induce Rapid Biosynthesis of Nitric Oxide (NO) in Arabidopsis thaliana Seedlings Plant Cell Physiol., March 1, 2006; 47(3): 346 - 354. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Lindermayr, G. Saalbach, G. Bahnweg, and J. Durner Differential Inhibition of Arabidopsis Methionine Adenosyltransferases by Protein S-Nitrosylation J. Biol. Chem., February 17, 2006; 281(7): 4285 - 4291. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. M. Crawford Mechanisms for nitric oxide synthesis in plants J. Exp. Bot., February 1, 2006; 57(3): 471 - 478. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Stohr and S. Stremlau Formation and possible roles of nitric oxide in plant roots J. Exp. Bot., February 1, 2006; 57(3): 463 - 470. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Perazzolli, M. C. Romero-Puertas, and M. Delledonne Modulation of nitric oxide bioactivity by plant haemoglobins J. Exp. Bot., February 1, 2006; 57(3): 479 - 488. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. A. J. Mur, T. L. W. Carver, and E. Prats NO way to live; the various roles of nitric oxide in plant-pathogen interactions J. Exp. Bot., February 1, 2006; 57(3): 489 - 505. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F.-Q. Guo and N. M. Crawford Arabidopsis Nitric Oxide Synthase1 Is Targeted to Mitochondria and Protects against Oxidative Damage and Dark-Induced Senescence PLANT CELL, December 1, 2005; 17(12): 3436 - 3450. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. U. IGAMBERDIEV, K. BARON, N. MANAC'H-LITTLE, M. STOIMENOVA, and R. D. HILL The Haemoglobin/Nitric Oxide Cycle: Involvement in Flooding Stress and Effects on Hormone Signalling Ann. Bot., September 1, 2005; 96(4): 557 - 564. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. A.J. Mur, I. E. Santosa, L. J.J. Laarhoven, N. J. Holton, F. J.M. Harren, and A. R. Smith Laser Photoacoustic Detection Allows in Planta Detection of Nitric Oxide in Tobacco following Challenge with Avirulent and Virulent Pseudomonas syringae Pathovars Plant Physiology, July 1, 2005; 138(3): 1247 - 1258. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Ohwaki, M. Kawagishi-Kobayashi, K. Wakasa, S. Fujihara, and T. Yoneyama Induction of Class-1 Non-symbiotic Hemoglobin Genes by Nitrate, Nitrite and Nitric Oxide in Cultured Rice Cells Plant Cell Physiol., February 1, 2005; 46(2): 324 - 331. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. U. Igamberdiev and R. D. Hill Nitrate, NO and haemoglobin in plant adaptation to hypoxia: an alternative to classic fermentation pathways J. Exp. Bot., December 1, 2004; 55(408): 2473 - 2482. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Sokolovski and M. R. Blatt Nitric Oxide Block of Outward-Rectifying K+ Channels Indicates Direct Control by Protein Nitrosylation in Guard Cells Plant Physiology, December 1, 2004; 136(4): 4275 - 4284. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Perazzolli, P. Dominici, M. C. Romero-Puertas, E. Zago, J. Zeier, M. Sonoda, C. Lamb, and M. Delledonne Arabidopsis Nonsymbiotic Hemoglobin AHb1 Modulates Nitric Oxide Bioactivity PLANT CELL, October 1, 2004; 16(10): 2785 - 2794. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. J. Corpas, J. B. Barroso, A. Carreras, M. Quiros, A. M. Leon, M. C. Romero-Puertas, F. J. Esteban, R. Valderrama, J. M. Palma, L. M. Sandalio, et al. Cellular and Subcellular Localization of Endogenous Nitric Oxide in Young and Senescent Pea Plants Plant Physiology, September 1, 2004; 136(1): 2722 - 2733. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Zeier, M. Delledonne, T. Mishina, E. Severi, M. Sonoda, and C. Lamb Genetic Elucidation of Nitric Oxide Signaling in Incompatible Plant-Pathogen Interactions Plant Physiology, September 1, 2004; 136(1): 2875 - 2886. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. F. Vanin, D. A. Svistunenko, V. D. Mikoyan, V. A. Serezhenkov, M. J. Fryer, N. R. Baker, and C. E. Cooper Endogenous Superoxide Production and the Nitrite/Nitrate Ratio Control the Concentration of Bioavailable Free Nitric Oxide in Leaves J. Biol. Chem., June 4, 2004; 279(23): 24100 - 24107. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Lillo, C. Meyer, U. S. Lea, F. Provan, and S. Oltedal Mechanism and importance of post-translational regulation of nitrate reductase J. Exp. Bot., June 1, 2004; 55(401): 1275 - 1282. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. C. Bethke, M. R. Badger, and R. L. Jones Apoplastic Synthesis of Nitric Oxide by Plant Tissues PLANT CELL, February 1, 2004; 16(2): 332 - 341. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Desikan, M.-K. Cheung, J. Bright, D. Henson, J. T. Hancock, and S. J. Neill ABA, hydrogen peroxide and nitric oxide signalling in stomatal guard cells J. Exp. Bot., January 2, 2004; 55(395): 205 - 212. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Garcia-Mata, R. Gay, S. Sokolovski, A. Hills, L. Lamattina, and M. R. Blatt Nitric oxide regulates K+ and Cl- channels in guard cells through a subset of abscisic acid-evoked signaling pathways PNAS, September 16, 2003; 100(19): 11116 - 11121. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Loque, P. Tillard, A. Gojon, and M. Lepetit Gene Expression of the NO3- Transporter NRT1.1 and the Nitrate Reductase NIA1 Is Repressed in Arabidopsis Roots by NO2-, the Product of NO3- Reduction Plant Physiology, June 1, 2003; 132(2): 958 - 967. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Desikan, R. Griffiths, J. Hancock, and S. Neill A new role for an old enzyme: Nitrate reductase-mediated nitric oxide generation is required for abscisic acid-induced stomatal closure in Arabidopsisthaliana PNAS, December 10, 2002; 99(25): 16314 - 16318. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. M. Kaiser, H. Weiner, A. Kandlbinder, C.-B. Tsai, P. Rockel, M. Sonoda, and E. Planchet Modulation of nitrate reductase: some new insights, an unusual case and a potentially important side reaction J. Exp. Bot., April 15, 2002; 53(370): 875 - 882. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||