Journal of Experimental Botany, Vol. 51, No. 347, pp. 1099-1105,
June 2000
© 2000 Oxford University Press
Nitrate reductases from leaves of Ricinus (Ricinus communis L.) and spinach (Spinacia oleracea L.) have different regulatory properties
Universität Würzburg, Lehrstuhl für Molekulare Pflanzenphysiologie und Biophysik, Julius-von-Sachs-Platz 2, D-97082 Würzburg, Germany
Received 30 September 1999; Accepted 2 February 2000
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Abstract |
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The activity of nitrate reductase (+Mg2+, NRact) in illuminated leaves from spinach, barley and pea was 50–80% of the maximum activity (+EDTA, NRmax). However, NR from leaves of Ricinus communis L. had a 10-fold lower NRact, while NRmax was similar to that in spinach leaves. The low NRact of Ricinus was independent of day-time and nitrate nutrition, and varied only slightly with leaf age. Possible factors in Ricinusextracts inhibiting NR were not found. NRact from Ricinus, unlike the spinach enzyme, was very low at pH 7.6, but much higher at more acidic pH with a distinct maximum at pH 6.5. NRmax had a broad pH response profile that was siilar for the spinach and the Ricinus enzyme. Accordingly, the Mg2+-sensitivity of NR from Ricinus was strongly pH-dependent (increasing sensitivity with increasing pH), and as a result, the apparent activation state of NR from a Ricinus extract varied dramatically with pH and Mg2+concentration. Following a light–dark transition, NRact from Ricinus decreased within 1 h by 40%, but this decrease was paralleled by NRmax. In contrast to the spinach enzyme, Ricinus-NR was hardly inactivated by incubating leaf extracts with ATP plus okadaic acid. A competition analysis with antibodies against the potential 14-3-3 binding site around ser 543 of the spinach enzyme revealed that Ricinus-NR containes the same site. Removal of 14-3-3 proteins from Ricinus-NR by anion exchange chromatography, activated spinach-NR but caused little if any activation of Ricinus-NR. It is suggested that Mg2+-inhibition of Ricinus-NR does not require 14-3-3 proteins. The rather slow changes in Ricinus-NR activity upon a light/dark transient may be mainly due to NR synthesis or degradation.
Key words: Activation state, nitrate reductase, Ricinus communis L., protein phosphorylation, 14-3-3 proteins.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Nitrate reductase (NR, EC 1.6.6.1) from spinach leaves is rapidly inactivated by serine phosphorylation and subsequent binding to 14-3-3 proteins in the presence of divalent cations (for review see Kaiser et al., 1999
). Therefore, NR activity in the presence of Mg2+ (NRact) usually reflects activity of dephospho-NR. In contrast, in buffer containing excess EDTA, all NR forms are active and the measured activity reflects the total amount of NR (=NRmax). The ratio of NRactx100/NRmax is termed the ‘activation state’. In leaves, the NR activation state is usually 1.5–3 times higher in the light than in the dark, and this has been observed for a number of higher plant species belonging to different genera, like Arabidopsis (Su et al., 1996), Brassica campestris (Kojima et al., 1995), Cucurbita pepo (Glaab and Kaiser, 1996; Lillo et al., 1997), Triticum aestivum L. (J Glaab and WM Kaiser, unpublished results), Pisum sativum L. (Glaab and Kaiser, 1993), Nicotiana tabacum (Nussaume et al., 1995; Scheible et al., 1997), Hordeum vulgare L. (Lillo et al., 1996a, Man et al., 2000), Zea mays L. (Huber et al., 1994; Merlo et al., 1995), and the CAM plant Bryophyllum fedtschenkoi (Lillo et al., 1996b). Thus, the regulation of NR and the extent and velocity of NR activity changes in leaves exposed to light–dark transitions are similar in many higher plant species. However, NR from leaves of Ricinus communis L. has unusual regulatory properties which are described below. |
Materials and methods |
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Plant material
Spinach (Spinacia oleracea L. cv. Polka F1) was grown in a greenhouse. The mean daylength was 11 h with supplementary illumination (HQi, 400 W; Schreder, Winterbach, Germany) at a total photon flux density of 250–400 µmol m-2 s-1 photosynthetically active radiation. Air humidity varied from 60–80%, and day/night temperature from 20–26 °C and 16–22 °C. The plants were fed with a commercial nitrate fertilizer. For experiments, leaves of 7–9-week-old spinach plants were used. Seeds of Ricinus (Ricinus communis L.) were germinated in vermiculite moistened with 0.5 mM CaSO4. After 12–13 d, seedlings were transferred to pots (one plant per pot) containing 4.0 l of well-aerated nutrient solution (10 mM KNO3, 1 mM CaCl2, 1.3 mM K2HPO4, 2.7 mM KH2PO4, 2 mM MgSO4, 0.2 mM NaFe-EDTA, and trace elements according to Johnson et al. (Johnson et al., 1957
). All nutrient solutions were replaced every second day. Plants were illuminated for 10 h during the day (HQI, 400 W; Schreder, Winterbach, Germany) at a total photon flux density of 320 µmol m-2 s-1 photosynthetically active radiation. Mean air temperature was 24 °C. Ricinus leaves were harvested 4–5 weeks after germination.
In vitro assay of NR activity
Leaf material was ground with liquid nitrogen and 3 ml of extraction buffer (100 mM HEPES, pH 7.6, 5 mM DTT, 10 µM FAD, 15 mM MgCl2, 2 mM Pefabloc [4-(2-aminomethyl)-benzene-sulphonylfluoride hydrochloride, 10 µM leupeptin, 0.1 mM PMSF (phenyl methylsulphonylfluoride), 0.02% casein, 0.5% polyvinylpolypyrolidone, and 0.05% BSA) was added to 1 g FW. After continuous grinding until thawing the suspension was centrifuged (14 500 g, 10 min, 4 °C). The supernatant was desalted on Sephadex G 25 spin columns (1.5 ml gel volume, 650 µl extract, 4 °C) equilibrated with the extraction buffer without the protease-inhibitors. ith aliquots of the supernatant the following assays were carried out:
- (a) Determination of NRact: Extract (250 µl (Ricinus) or 100 µl (spinach)) was added to a mixture of 750 µl (Ricinus) or 900 µl (spinach) buffer (50 mM HEPES, pH 7.6, 5 mM DTT, 10 µM FAD, 15 mM MgCl2, 5 mM KNO3, and 0.2 mM NADH). The reaction was carried out at 24 °C. After 5 min the reaction was stopped by adding 125 µl zinc acetate (0.5 M).
- (b) Determination of NRmax: If not mentioned otherwise, extracts of 250 µl (Ricinus) or 100 µl (spinach) were preincubated at 24 °C with 20 mM EDTA, 5 mM AMP and 5 mM
(final concentrations). AMP+Pi accelerate activation, in addition to EDTA (Athwal et al., 1998). After 13 min, buffer (50 mM HEPES, pH 7.6, 5 mM DTT,10 µM FAD, and 15 mM EDTA) was added to a final volume of 1 ml. After 2 min (total preincubation time: 15 min) the reaction was started by adding 5 mM KNO3 and 0.2 mM NADH. Five minutes later the reaction was stopped by adding 125 µl zinc acetate (0.5 M). Excess NADH was removed by phenazine methosulphate (PMS) treatment. The colorimetric determination of nitrite formed additionally by NR during reaction assay was carried out as described previously (Hageman and Reed, 1980).
- (b) Determination of NRmax: If not mentioned otherwise, extracts of 250 µl (Ricinus) or 100 µl (spinach) were preincubated at 24 °C with 20 mM EDTA, 5 mM AMP and 5 mM
Partial purification of NR
NR was prepared via fractionation by PEG-8000 and anion-exchange chromatography on a Resource Q column (Pharmacia, Heidelberg, Germany) using a Pharmacia/LKB fast protein liquid chromatography system. All enzyme purification steps were performed at 0–4 °C as rapidly as possible. About 15 g of Ricinus leaves or spinach leaves were harvested 2 h after the beginning of the light phase and ground in liquid nitrogen; 45 ml of extraction buffer containing 100 mM HEPES, pH 7.6, 5 mM DTT, 10 µM FAD, 15 mM MgCl2, 2 mM Pefabloc, 10 µM leupeptin, 0.1 mM PMSF, and 0.5% polyvinylpolypyrrolidone was added and grinding was continued until thawing. After centrifugation (15 000 g, 20 min, 4 °C) the cleared supernatant was subjected to a 4–2% (w/v) PEG-8000 precipitation. The precipitated protein was collected by centrifugation at 15 000 g for 10 min (4 °C) and suspended in 8 ml of MOPS buffer (50 mM MOPS pH 7.5, 1 mM DTT, 10 mM MgCl2, 1 mM benzamidine, and 1 mM µ-amino-n-caproic acid). After centrifugation at 12 000 g for 10 min to remove the aggregated insoluble proteins, the supernatant (protein content 40 mg) was loaded onto a 1 ml Resource Q column. Protein was eluted with a 35 ml linear gradient from 0–500 mM NaCl in MOPS buffer. The eluate was collected in 1 ml fractions. The fractions were examined for NR activity and protein and for 14-3-3 proteins.
Protein determination
The protein content of the samples was determined with BCA reagent (Pierce, Rockford, Ill., USA) and BSA as a standard.
Western blotting
After electrophoresis in 10% (NR) to 14% (14-3-3 protein) SDS polyacrylamide gels (Laemmli, 1970) the proteins were transferred onto nitrocellulose membranes. The protein blots were immunostained with serum against NR or against 14-3-3 proteins. NR antibodies were raised in rabbits. The NR antigen was prepared by coupling a synthetic peptide (corresponding to residues 545–551 of spinach-NR) to partially purified protein derivative of tuberculin according to Weiner (Weiner, 1995). Antibodies to 14-3-3s were a gift from C MacKintosh (Dundee University, Scotland). These antibodies were raised in sheep with purified 14-3-3s from spinach leaves as antigen. The antigen/primary antibody complex was visualized by alkaline phosphatase-linked IgGs and p-nitro-blue tetrazolium chloride/ 5-bromo-4-chloro-indolyl-phosphate.
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Results |
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Typical NR activation states in different plant species
While in spinach, pea and barley (and other plant species not shown here), the activation state in the light was between 60% and 88%, it was always below 10% in R. communis (Table 1
).
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As NRmax and NRact in leaves are not constant during the day, but may vary diurnally (Scheible et al., 1997
; Man et al., 2000), a complete diurnal time-course for NRmax and NRact was measured in Ricinus and, for comparison in spinach (Fig. 1). Again, Ricinus-NR did not reach an activation state comparable to that in spinach at any time during the day. As in other plants, NRmax was low during the night and increased during the first half of the day, with a significant decrease during the second half. Western blotting and subsequent density scanning confirmed the results of the NRmax measurement (not shown).
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The low activation state of Ricinus-NR did not depend much on leaf age, although NRmax varied considerably (Table 2
). The highest activation state obtained was 13%, which is still much lower than in all other plants investigated so far.
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The low activation state of Ricinus-NR was also unaffected by nitrate availibility in the nutrient solution (Table 3
). When external nitrate was increased from 1 mM to 10 mM, the nitrate content of the leaves was elevated from about 1 µmol g-1 FW to about 20 µmol g-1 FW. This was paralleled by an increase of NRmax from about 4 µmol g-1 FW h-1 (light) to about 20 µmol g-1 FW h-1. However, in spite of that large difference in NRmax, the activation state in the light at ambient CO2 rarely exceeded 4%.
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Ricinus leaves do not contain factors that inhibit NR in crude extracts
A possible explanation for the very low NRact in Ricinus might be the presence of some factors in Ricinus leaves that inhibit NR after extraction. To test this, NRact and NRmax were determined in extracts from illuminated Ricinus and spinach leaves, and in mixtures (1 : 1, v/v) thereof. As shown in Table 4, the NR activity in the mixture almost matched the sum of NR activities in the single extracts. Thus, it is unlikely that Ricinus extracts contained unknown factors inhibitory for NR.
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The activity of NRs from Ricinus and from spinach have different sensitivity to pH and Mg2+
It has been shown that artifical acidification of leaf or root tissues activates NR (Kaiser and Brendle-Behnisch, 1995). Other activating conditions such as anoxia or respiratory inhibitors may also act via cytosolic acidification (Kaiser et al., 1999). However, in the standard extraction and reaction buffers used here, the pH was 7.6. The cytosolic pH in vivo may different, especially under extreme conditions like anoxia. Therefore the pH-response of NR from Ricinus and from spinach was compared in desalted crude extracts (Fig. 2). For NRmax both pH-response curves were similar, with a broad optimum between pH 7 and pH 8. However, the curves for NRact (10 mM Mg2+) of Ricinus and spinach were different. While NRact from spinach had a broad optimum between 6.5 and pH 7.5, NRact from Ricinus had a distinct optimum around pH 6.5, and was very low at pH values above 7.3. Accordingly, the apparent activation state of Ricinus-NR, unlike NR from spinach, was strongly pH-dependent within the range from pH 7.6 to pH 6.5. At pH 7.6, Ricinus-NR was much more sensitive to Mg2+ inhibition than the spinach enzyme. The apparent I50 (Mg2+) for spinach-NR was about 5 mM (Fig. 3). For Ricinus-NR, the exact I50 could not be determined, since in the crude, desalted extract Ricinus-NR was already inhibited to less than 50% when Mg2+ was omitted from the reaction medium, and was almost completely inhibited with 10 mM Mg2+. At pH 6.8, Ricinus-NR, unlike spinach-NR, was much less inhibited with Mg2+ than at pH 7.6.
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Ricinus extracts contain 14-3-3 proteins which are removed during partial purification of NR
According to the multiple functions of 14-3-3s in eukaryotic organisms it appeared rather improbable that Ricinus would not contain 14-3-3 proteins. However, in order to confirm this and also in order to see whether 14-3-3s were removed during partial purification of NR from Ricinus and from spinach, crude leaf extracts from both plants were purified as described in Materials and methods. Fractions were subjected to SDS-electrophoresis, blotted and immunodecorated with antibodies against NR and against 14-3-3s (Fig. 4, compare legend). Both extracts contained 14-3-3s which were eluted mainly in fractions 23–25, and were largely separated from NR fractions.
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The low activation state of Ricinus-NR above pH 7.3 (10 mM Mg2+) remained unchanged after removal of 14-3-3 proteins from NR by anion exchange chromatography (Table 5
), and the pH-profile was not changed (not shown). In contrast, when spinach leaf extracts were subjected to the same procedure, NR was activated (Table 5).
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Changes of NRact, NRmax and NR activation state in spinach and Ricinus leaves were compared during a light-dark transition (Fig. 5
). Here, Ricinus-NR was measured at pH 6.8 in order to have a higher activity than at pH 7.6. As expected, NRact of spinach decreased rapidly upon darkening, whereas NRact from Ricinus decreased only slowly and in parallel with NRmax. Accordingly, there was only a slow and minor decrease in the NR activation state in Ricinus in the dark.
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Modulation of NR in vitro
NR from spinach and from other plants can be rapidly inactivated in vitro by preincubation with ATP (+Mg2+, pH 7.6). Table 6 compares the inactivation by ATP of NR from spinach and Ricinus. For reasons mentioned above, activity measurements were not only carried out at the standard pH (7.6), but in addition at pH 6.8. While NRact from spinach was strongly decreased by preincubation with ATP, there was hardly any response of NR from Ricinus.
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Does Ricinus-NR have a 14-3-3 binding phosphorylation site?
To test whether Ricinus-NR has a 14-3-3-binding phosphorylation site like the spinach enzyme, the immunoreactivity of spinach-NR was characterized compared with Ricinus-NR on Western blots. Peptide antibodies were used that were directed to the sequence around serine 543 of the spinach enzyme. As shown in Fig. 6 such antibodies cross-reacted with both spinach and Ricinus-NR. The antibodies were obviously directed against the middle part of the antigenic peptide, as the ends of the peptide, unlike the middle part, did not prevent the immunodecoration of NRs. Importantly, the serine residue corresponding to serine 543 of spinach-NR seemed to be essential for peptide binding to the antibodies. So, Ricinus-NR seems to have the same potential 14-3-3 binding site as the spinach enzyme.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The activation state of NR from Ricinus leaves was generally much lower than in all other plants examined so far, if determined under the widely used standard assay conditions (high Mg2+ and pH 7.6). Then, NRact of Ricinus was between 0.1 and 1 µmol g-1 FW h-1. NRact in vivo would be somewhat higher (1–3 µmol g-1 FW h-1, assuming a cytosolic pH around 7.2 and cytosolic free Mg2+ in the low millimolar range. For Ricinus plants grown on 4 mM
, mean nitrate assimilation rates in the leaves over a period of 10 d have been estimated as about 1 µmol g-1 FW h-1, if nitrate reduction was restricted to the light phase only (Peuke et al., 1996). Thus, at least under substrate saturation, even the above low NRact would be sufficient to support growth. These data suggest that Ricinus-NR has regulatory properties different from spinach or other plants. NR from Ricinus, unlike the spinach enzyme, was neither activated by preincubation with EDTA plus AMP, nor inactivated with ATP. Further, unlike spinach-NR, Ricinus-NR was still inactive in partially purified and largely 14-3-3-free preparations. Thus, while Ricinus-NR appears to have a potential 14-3-3 binding site like the spinach enzyme, the inhibition of Ricinus-NR by Mg2+ does not require 14-3-3 proteins.
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Acknowledgments |
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This work was supported in part by the DFG, SFB 251, and by EU project BIO4-CT97-2231. The skilled technical assistance of Maria Lesch is gratefully acknowledged. We also thank C MacKintosh (Dundee) for a generous gift of serum against spinach 14-3-3s.
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Notes |
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1 To whom correspondence should be addressed. Fax: +49 931 888 6158. E-mail: kaiser{at}botanik.uni\|[hyphen]\|wuerzburg.de
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Abbreviations |
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FW, fresh weight; NR, NADH-nitrate reductase; NRact, actual NR activity; NRmax, maximum NR activity..
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