Plant Physiol. (1 995) 107: 225-231
Circadian Oscillation of Nitrate Reductase Activity in Gonyaulaxpolyedra 1s Due to Changes in CeIIuIar Protein LeveIs' Camila Balsamo Ramalho, J.Woodland Hastings*, and Pio Colepicolo Universidade de São Paulo, Instituto de Química, Departamento de Bioquímica, São Paulo, CP 20780, Brazil (C.B.R., P.C.); and Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, Cambridge, M A 021 38-2020 (J.W.H.) controlled proteins have been isolated and fully characterized from any system with regard to their mechanism of regulation. We describe in this paper circadian regulation of another Gonyuulux protein, NR, the first enzyme of the nitrate assimilatory pathway, catalyzing the reduction of nitrate to nitrite (Redinbaugh and Campbell, 1991; Warner and Kleinhofs, 1992). The first evidence that NR activity exhibits a circadian oscillation in Gonyuulux was reported by Harris (1975), which we have confirmed. The present studies have shown that the amount of the NR protein changes; in cell extracts it is abundant during the day phase but absent in the night extracts.
A circadian rhythm in the activity of nitrate reductase (NR; EC 1.6.6.1) isolatedfrom the marine dinoflagellate Gonyaulaxpolyedra is shown to be attributable to the daily synthesis and destruction of the protein. l h e enzyme was purified in three steps: gel filtration on S-300 Sephacryl, an Affigel-Blue column, and a diethylaminoethyl ion-exchange column. Undenatured protein shows a molecular mass of about 31O kD; based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the enzyme appears to be composed of six possibly identical subunits. The amino acid composition of the G. polyedra NR is very similar to that reported for the NR of barley leaves, Chlorella vulgaris, and Ankistrodesmusbraunii. l h e experiments reported indicate that the cellular expression of N R is under circadian control. In extracts of cells grown under either constant dim light or a light-dark cycle, the activity of NR exhibits a daily rhythm, peaking at midday phase, as does photosynthesis. Staining with affinity-purified polyclonal antibodies, raised in rabbits against purified NR, shows that the amount of protein changes by a factor of about 10, with the maximum occurring in midday phase.
MATERIALS A N D METHODS Cell Culture
Cultures of the dinoflagellate Gonyuulux polyedru were grown in f/2 medium (Guillard and Ryther, 1962) under alternating periods of 12 h of light (cool-white fluorescent; 150 p E m-' s-') and 12 h of dark (LD 12:12) to a cell concentration of about 104cells/mL (Dunlap and Hastings, 1981). For experiments under constant conditions, cells were maintained under LL (35 pE m-* s-I). Cells were harvested in the middle of the light period by filtration on Whatman (Clifton, NJ) 541 filter paper. The beginning of the light period is defined as time zero (LD O), and for cells in constant conditions, it is circadian zero time (CT O).
Eukaryotic organisms, including humans, possess an interna1 daily biological clock; its most fundamental function is to control the time of day at which different processes occur (Sweeney, 1987; Hastings et al., 1991; Takahashi, 1991). In the unicellular alga Gonyuulux polyedru numerous processes are clock controlled: bioluminescence occurs during night phase, cell aggregation and photosynthesis peak during the day, and cell division occurs at the transition between night and day (Johnson and Hastings, 1986). The circadian changes in bioluminescence are correlated with daily changes in the cellular amounts of both luciferase and luciferin (substrate) binding protein, and there is a daily pulse in the synthesis of luciferin-binding protein that is regulated at the translational leve1 (Morse et al., 1989; Milos et al., 1990; Lee et al., 1993). Other than the proteins responsible for the bioluminescence, no circadian-
Purification and Assay of N R
Cell extracts were prepared by harvesting 15 L of cultures at midday, when NR activity is highest. Freshly harvested cells were resuspended in 20 mL of extraction buffer (0.05 M Tris-HC1 buffer, pH 7.9, containing 0.05 M SUC,5 mM 2-mercaptoethanol, and 0.01 M EDTA) and broken by high-pressure N, (2500 psi). Cell debris was removed by centrifugation at 15,OOOg for 15 min. The clarified crude extract was loaded on a 1.5- X 40-cm S-300 Sephacryl (Bio-Rad) column that had been equilibrated with extract buffer and calibrated with molecular
Supported in part by the National Institutes of Health (GM18546), the Office of Naval Research (N00014-94-10575), International Foundation for Science, Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, and Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP). C.B.R. was the recipient of a research fellowship from FAPESP. * Corresponding author; e-mail
[email protected]. edu; fax 1-617-495-9300.
Abbreviations: AbNR, antibody to nitrate reductase; CT, circadian time; DD, constant darkness; LD, light-dark cycle, 12 h light-12 h dark; LL, constant dim light; NR, nitrate reductase. 225
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mass standards (ferritin, 430 kD; aldolase, 158 kD; and Cyt
c, 12.3 kD; Boehringer Mannheim) and dextran blue (Sigma). The fractions containing activity were pooled and loaded onto a 2- X 5-cm Affigel-Blue column (Bio-Rad) equilibrated with extraction buffer and washed exhaustively with the same buffer. The washings were discarded and NR was eluted with a gradient of NADH. Fractions containing NR activity were pooled and loaded on a 2- x 3-cm coarse DEAE-cellulose column and washed with extraction buffer. A gradient from O to 0.5 M NaCl in extraction buffer was used to elute NR, and the active fractions were stored at -70°C after desalting. Protein content was estimated in the eluted fractions by A,,, and determined quantitatively by reaction either with the Folin reagent (Lowry et al., 1951) or by the Coomassie blue dye-binding assay (Bradford, 1976). Egg albumin (Sigma) was used as the standard protein for calibration curves. For the NR assays, the reaction mixtures contained 0.2 M phosphate buffer, pH 7.7, 10 mM KNO,, 1 mM 2-mercaptoethanol, 0.5 mL of the cell extract, and 40 mM NADH to start the reaction, with incubation at 25°C for 10 to 60 min (depending on the enzyme activity). The reaction was stopped by boiling, and nitrite concentration was determined by adding 1 mL of 60 mM sulfanilamide solution followed by 1 mL of 4 mM n-(1-naphthyl) ethylenediamide dihydrochloride solution. Thus, the red color (measured at 543 nm), which develops after 10 min, is proportional to nitrite concentration. One unit of NR is defined as the amount of enzyme required to produce 1 pmol of nitrite/ min at 25°C.
Plant Physiol. Vol. 107, 1995
scribed above. Controls containing preimmune serum were assayed under the same conditions. RESULTS Purification of NR
Since Blue-Sepharose binds NR from higher plants and algae (Campbell and Smarelli, 1978),we purified Gonyaulax NR utilizing the same concept, with a few mcdifications. The active fractions from the S-300 Sephacryl column (Fig. 1) were pooled and loaded on a Affigel-Blue column (Fig. 2A). The enzyme was then eluted with a gradient of NADH, immediately loaded onto a DEAE-cellulose column (Fig. 2B), and eluted with a gradient of NaCl. The proceclure resulted in a protein purified almost 200fold, with a recovery of about 30%,as summarized in Table I. Based on SDS-PAGE (Fig. 31, the protein It the final stages of purification was estimated to be about 90% pure and to have ,I subunit molecular mass of about 52 kD. The apparent molecular mass of the undenatured IVR was determined by gel filtration chromatography on a calibrated S-300 column to be about 310 kD (Fig. 11, meaning that the native protein could be a hexamer of approximately 52 kD, corresponding to the SDS-PAGE value. One advaiitage of NR purification by the 4ffigel-Blue method, in contrast to multistep procedures (MacGregor et al., 1974; Solomonson et al., 1975; Campbell and Wray, 1983; Horner, 1983), is that a high specific activity NR can be obtained in good yield within 3 to 4 h aftei- extraction. Properties
Electrophoresis and Western Blotting
SDS-PAGE was performed on 10% gels according to the method of Laemmli (1970). The gels were stained with Coomassie blue or were transferred to a nitrocellulose membrane for western blots (Towbin et al., 19791, using protein A labeled with lz5I (New England Nuclear) and autoradiography for detection of bound antibody. The molecular mass standards (Sigma) were phosphorylase B, 97 kD; BSA, 66 kD; egg albumin, 45 kD; lactic dehydrogenase, 36 kD; carbonic anhydrase, 29 kD; and trypsinogen, 24 kD.
Antibody Preparation
Severa1 milligrams of purified NR in 1.0 mL were emulsified by sonication with 1 volume of complete Freund's adjuvant and injected subcutaneously into rabbits. A booster injection of 3 mg of NR, emulsified with the incomplete Freud's adjuvant, was given after 1 month, and the animals were bled 2 weeks later. Affinity-purified antibody was eluted with 0.2 M Gly, pH 2.8, from anti-NR serumtreated nitrocellulose strips to which purified NR had been transferred after SDS-PAGE (Olmsted, 1981). Before use, it was dialyzed against 10 mM Tris-buffered saline, pH 7.2. For the antibody titration, samples of crude extracts containing 3 units of NR were incubated at 4°C with different amounts of NR antibody. The activity remaining after 1 h, when the reaction was complete, was measured as de-
The properties of Affigel-Blue-purified NR were found to be somewhat different from those reported for NR from other species (Adams and Mortenson, 1985). The Michaelis-Menten constant using nitrate as stibstrate was found to be 250 p ~ with , NADPH it was 35 PM, and with NADH it was 80 FM. The assays were perforried in 0.2 M vo
430 kDa
158 kDa
12,5 kDa
1 ,l. 1 . ,l. I 0.6
0.3
30
40
50
60
Volume, ml Figure 1. Cel filtration of NR. A crude extract of Gonyaulax cells harvested during the day was chromatographed on a Sephacryl S-300 column. The NR activity of each fraction was measLred (ordinate). Activity peaked at about 31 O kD; the standards are noted by arrows. Vo, Void volume.
227
Circadian Rhythm in the Amount of Nitrate Reductase in Conyaulax
100
200
300
-
300
Table I. Purification of NR from Conyaulax Flow sheet for the activities and yields of NR at the sequential purification steps, starting with crude extract from 15 L of a dayphase culture. Peak fractions were pooled at each step, and one unit of NR is defined as the amount of enzyme required to produce 1 jumol of nitrite/min at 25°C. Protein was measured according to the method of Bradford (1976).
0.30 -
0.20 •
Step
O
0.10 '
CD
I
o 12
B/
tcu
0.10
Crude extract Centrifuged Sephacryl S-300 Affigel-Blue DEAE
Total Activity 3 units 9.6 9.1 5.7
3.7 2.3
Proteinb
Specific Activity
mg
units/mg 0.023 0.028 0.056
410 325 102 0.84
4.46
4.25
0.54
Recovery
%
100 95 60 39 24
a
Pooled peak fractions. One unit of NR is defined as the amount of enzyme required to produce 1 ^imol of nitrite/min at 25°C. b Protein was measured according to Bradford (1976).
Antibody Inactivation of Enzyme
hI •o 0.05
o
L 0
50
Polyclonal antibodies against the purified NR were raised in rabbit and affinity purified. The antisera obtained inhibited NR activity very effectively, as shown in Figure 4. One microliter of affinity-purified AbNR inhibits more than 50% of total activity of NR after 1 h of incubation at 4°C. On western blots the antiserum reacts with a single band (about 52 kD; Fig. 3, lane 2). Preimmune serum had no inhibitory properties and showed no bands recognized on the western blots (data not shown).
100
Volume, ml
Circadian Changes in Activities and Amounts of Extractable NR
Figure 2. Purification of NR. A, Affigel-Blue chromatography. The crude extract was loaded on the column and washed with 10 volumes of extraction buffer. NR was eluted with a gradient of NADH (0-150 /J.M, dashed line), started at the time indicated by the arrow. B, DEAE-cellulose chromatography. Fractions containing NR activity from the Affigel-Blue column were pooled and loaded onto a DEAEcellulose column. After the column was washed with extraction buffer, NR was eluted by a gradient of NaCI (0-0.5 M; dashed line).
In cells grown under an LD, the NR activity in extracts made at different times exhibits a diurnal rhythm (Fig. 5A). This rhythm persists under LL (Fig. 5B) and may thus be classed as circadian. These data were obtained from cells Mw, kDa
B
phosphate buffer, pH 7,7, at 25°C. The enzyme has a much lower affinity for nitrate than for NAD(P)H, as do many other NRs described.
Amino Acid Composition The amino acid composition of the Gonyaulax NR subunit protein was similar in many respects to compositions reported for NR of the algae Chlorella and Ankistrodesmus, as well as barley (Table II). All of these proteins have higher levels of acidic amino acid residues (19.8 mol.% of Asn and Glu) than basic residues (14.4 mol % of Lys and Arg). The Gonyaulax NR showed a higher mol % of Met and Lys but less Thr and His than the other enzymes. The Ankistrodesmus enzyme had a relatively low mol % of Met, whereas the Chlorella enzyme had a slightly higher mol % of Pro and lower Arg than both Gonyaulax and Ankistrodesmus NR.
Figure 3. SDS-PAGE of the purified Gonyaulax NR visualized by Coomassie blue SDS-PAGE (A) and immunoblotting (B). NR was subjected to an SDS-PAGE and the proteins were stained with Coomassie blue.The proteins were also transferred electrophoretically to a nitrocellulose membrane and incubated with affinity-purified NR antibody and then 125l-protein A. The positions of molecular mass markers are indicated to the left of the figure.
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Table I I . Amino acid compositions o f NRs from various sources Amino acid composition of NR from Gonyaulax compared with those from the algae Chlorella (Giri and Ramadoss, 1979) and Ankistrodesmus (De Ia Rosa et al., 1981) and from barley (Kuo et al., 1982). The barley NR composition was determined with the protein subunit and data are given as mol%; n.d., not determined. Residues Convaulax Chlorella Ankistrodesmus Barlev ASP Thr Ser Glu Pro GlY Ala Val Met Ile Leu TYr Phe His LYS
Arg Half-cystine TrP
9.00 2.60 5.40 10.8 5.60 1 1 .o0 10.90 7.20 3.30 4.1 O 8.50 2 .o0 3.90 1.50 9.30 5.10 n.d. n.d.
9.32 6.40 4.78 10.50 8.41 9.38 11.10 7.07 1.93 4.34 7.54 2.06 3.1 8 2.93 4.95 3.76 2.33
9.86 6.1 3 6.31 9.01 6.24 8.94 9.96 7.30 0.25 4.72 7.87 3.60 4.08 2.55 5.1 8 5.92 2.06
n.d.
n.d.
10.33 5.72 6.37 12.38 6.39 9.1 O 7.75 6.78 1.68 4.40 7.62 2.63 3.10 2.62 5.91 5.39 n.d. n.d.
harvested, extracted, and assayed as described above every 2 h during a 24-h time interval. The peak NR specific activity is about 4 times higher in the day phase under both LD and LL regimes. Similar measurements were repeated six times with similar results, including the amplitude. Other workers in this laboratory have also obtained similar results. Crude extracts (20 pg protein/pL), prepared every 2 h from cells grown under both LD and LL conditions, were subjected to 10% SDS-PAGE, transferred to a nitrocellulose membrane, and probed with affinity-purified AbNR (Fig. 6). The stained bands show that in both cases the amount of NR protein present in crude extracts is higher in cells extracted during the day phase. This experiment was repeated three times with qualitatively similar results but with amplitude differences ranging from a factor of 3 to 10. DISCUSSION
Plant Physiol. Vol. 107, 1995
The Chlorella vulgaris NR is composed of f0u.r identical 90-kD subunits (Solomonson et al., 1975; Giri and Ramadoss, 1979), whereas the Ankistrodesmus braunii enzyme is composed of eight identical 60-kD subunits (De la Rosa et al., 1981). NRs isolated from Neurospora crassa and Rhodotorula glutinis are homodimers consisting 'Df two subunits of 115 kD (Pan and Nason, 1978) and l l e kD (Guerrero and Gutierrez, 1977), respectively. With regard to higher plants, reports of multiple, lower molwular mass subunits for NR from spinach and barley were subsequently shown to be a consequence of proteolytic "nicking" (Campbell and Wray, 1983; Fiddo and'Nctton, 1984). In vitro translation products of NR mRNA appear to be in this same molecular mass range (Cheng et al., 1986; Commere et al., 1986; Crawford et al., 1986). Based on the experiments described here, we propose that the Gonyaulax NR (320 kD) is composed of six identical subunits of 52-kD molecular mass. Given their different quaternary structures, the close similarities in the amino acid compositions for NRs isolated from Gonyaulax, Chlorella, Ankistrodesmus, and barley are striking. This is presumably related to the existence of a high degree of homology among these eukaryotic NRs; our analysis was carried out using the holoenzyme, which was also the case for the other two algal NRs. The inolybdenum co-factor, a presumed constituent of known eukaryotic NR complexes, must havc, a minimal contribution to the amino acid composition of the complex. Pan and Nason (1978) concluded that the ntolybdenum co-factor in N. crassa was a small peptide of lejs than 1000 D. If so, the contribution of the molybdenum co-factor peptide to the total amino acid composition of NR would be expected to be small. Based on the composition analysis, we conclude that Gonyaulax NR is an acidic protein with an amino acid composition very similar to that of two other algal and barley NRs. As for many other NRs (Adams and Mortenson, 1985), Gonyaulax NR is active at a neutra1 pH. The apparent K , values fountl are close to those described for the alga A.
2
Y
The major source of nitrogen in the marine ecosystem is in the form of nitrate (Adams and Mortenson, 1985), which is reduced to ammonia or amine for the biosynthesis of nitrogen-containing constituents (Solomonson and Barber, 1990; Crawford and Arst, 1993).The reduction of nitrate to nitrite is catalyzed by NR, which may be rate limiting in the nitrate assimilation process. NR is a high molecular weight flavoprotein that contains two enzymatic centers and uses NADH or NADPH as the electron donor. Although the overall enzymatic functions of NRs from various different eukaryotes are similar, and they are typically oligomeric enzymes, the number, sizes, and types of subunits can vary considerably (Hewitt, 1975; Guerrero et al., 1981; Kuo et al., 1982; Solomonson and Barber, 1990).
*
.CI .CI
Y
2
1.0
2.0
3.0
4.0
5.0
AbNR, pL Figure 4. Inactivation of NR activity by polyclonal anti-NR. Crude extracts containing 3 units of NR were incubated with different amounts of affinity-purified AbNR for 1 h at 4°C and the remaining activity was determined (ordinate).
Circadian Rhythm in the Amount of Nitrate Reductase in Conyaulax
229
Figure 5. Circadian changes in the activity of NR in extracts of cells harvested every 2 h. Cultures were kept in ID (A) and in LL (B), starting at the end of a dark period. The assays for NR activity were performed immediately after the cell disruption. CTs 8 and 23 h are noted by arrows.
6
12
18
26
Time, Hours
32
38
44
JO
Hours In Constant Light
braunii (De la Rosa et al., 1980) and for the fungus N. crassa (Greenbaun et al., 1978; Pan and Nason, 1978; Horner, 1983). The regulation of NR has been studied in numerous different species, both with respect to transcriptional expression and enzymatic activity. NR is known to be regulated by environmental factors, such as nitrate (Redinbaugh and Campbell, 1991) and light (Lillo, 1994), and phytochrome-mediated effects have been demonstrated in some systems. It may also be regulated by endogenous factors, including the circadian clock (Lillo, 1984; Lillo and Ruoff, 1989; Deng et al., 1990, 1991; Cheng et al., 1991; Pilgrim et al., 1993). In this last study, Arabidopsis NR mRNA accumulation exhibited a circadian oscillation that persisted for at least 5 d in both LL and DD. Enzyme activity also appeared to exhibit circadian changes, but it was far less robust. Cheng et al. (1991) recorded a single cycle of message expression under such conditions, also in Arabidopsis. Deng et al. (1990, 1991) reported a robust rhythm of tobacco NR mRNA, and also measured both NR protein and activity in extracts of plants transferred to LL and DD, interpreting single peaks as being indicative of a circadian rhythm. In the study of Lillo (1984) of barley NR, a circadian rhythm of enzyme activity was demonstrated to persist for up to 3 d, albeit with a rather small amplitude. In a later study of corn, Lillo and Ruoff (1989) reported that the onset of light in an LD induced a spike (approximately 2 h) in NR mRNA accumulation, followed by a slow increase and decrease in the NR protein and activity. They modeled the
persistent circadian oscillations of NR in terms of positive and negative feedback loops. In extracts of Gonyaulax cells grown under a daily LD, the enzymatic activity of NR cycles with an amplitude somewhat greater than that reported for other systems: midday phase is about 4 times greater than at the minimum. This rhythm continues in cells maintained in LL, indicative of circadian regulation. Using western blot techniques we found that the extractable levels of NR protein change in parallel with its activity, with an amplitude that is equal to or even greater than the in vivo rhythm. This indicates that the protein is actually synthesized and destroyed each day, as opposed to what might seem a more economical alternative to the same end, such as inhibition and activation, by phosphorylation, for example. This latter possibility is judged not to be likely in this case, since polyclonal antibody, which should recognize a modified protein, was used in the western blots. Synthesis and destruction has previously been demonstrated for two other circadian-regulated Gonyaulax proteins (luciferase and luciferin-binding protein), but these differ from NR in that they are present and active during the night phase and absent by day. Thus, NR represents a protein whose synthesis and abundance is 180° out of phase with the others studied, providing the possibility for determining the molecular basis for the determination of phase in circadian expression. In quite a number of other systems (see Kloppstech, 1985; Loros et al., 1989; Kay, 1993; Piechulla, 1993), there are
B LIGHT
DARK
DIM LIGHT
97 66 45 36 29 24
Figure 6. Oscillation in the amount of the NR protein. Cells maintained under LD (A) and LL (B) were harvested every 2 h during a 24-h period, beginning at dawn (A) and at subjective dawn (B). Equal amounts of soluble proteins were loaded onto the SDS-PAGE and transferred electrophoretically onto a nitrocellulose membrane, which was treated with affinity-purified AbNR and then 125l-protein A. The two panels are autoradiographs of the membranes spanning a 24-h time in both cases; a circadian rhythm in immunologically reactive protein is observed.
Ramalho et al.
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circadian r h y t h m s of mRNA accumulation, similar t o t h e NR examples cited above. For at least some genes t h e Gonyaulax system appears t o be different, i n that message
levels remain constant, whereas protein synthesis, which is confined t o a specific time of d a y for luciferin-binding protein, is regulated translationally (Morse e t al., 1989). O t h e r proteins in Gonyaulax may be similarly controlled. Milos e t al. (1990) showed by pulse labeling i n vivo and two-dimensional gel analysis of the extracted proteins that m a n y a r e synthesized preferentially d u r i n g either t h e day or night phase. They provided evidence that those proteins a r e also under translational control: in vitro translation of mRNAs for the proteins were similar in rate w i t h messages isolated at different times of day. It will be interesting to determine w h e t h e r NR in Gonyaulax is also regulated translationally . ACKNOWLEDCMENTS We are grateful to Dr. David Morse (University of Montreal) and to Dr. Lawrence Fritz (Northern Arizona State University) for valuable suggestions. Received August 10, 1994; accepted September 29,1994. Copyright Clearance Center: 0032-0889/95/ 107/0225/07
LITERATURE CITED Adams MWW, Mortenson LE (1985) Mo reductases: nitrate reductase and formate dehydrogenase. In TG Spiro, ed, Molybdenum Enzymes. John Wiley & Sons, New York, pp 519-593 Bradford MMA (1976) A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Ana1 Biochem 7 2 248-254 Campbell JM, Wray JL (1983) Purification of barley nitrate reductase and demonstration of nicked subunits. Phytochemistry 22: 2375-2382 Campbell WH, Smarelli J (1978) Purification and kinetics of higher plant NADH:nitrate reductase. Plant Physiol 61: 611-616 Cheng C-L, Acedo GN, Dewdney J, Goodman HM, Conkling, MA (1991) Differential expression of the two Arabidopsis nitrate reductase genes. Plant Physiol96 275-279 Cheng C-L, Dewdney J, Kleinhofs A, Goodman HM (1986) Cloning and nitrate induction of nitrate reductase mRNA. Proc Natl Acad Sci USA 8 3 6825-6828 Commere B, Cherel I, Kronenberger J, Galanhan F, Caboche M (1986) In vitro translation of nitrate reductase messenger RNA from maize and tobacco and detection with an antibody directed against the enzyme of maize. Plant Sci 4 4 191-203 Crawford NM, Arst HN Jr (1993) The molecular genetics of nitrate assimilation in fungi and plants. Annu Rev Genet 27: 115-146 Crawford NM, Campbell WH, Davis RW (1986)Nitrate reductase from squash: cDNA cloning and nitrate regulation. Proc Natl Acad Sci USA 83: 8073-8076 De la Rosa MA, Diez J, Vega JM, Losada M (1980) Purification and properties of assimilatory NAD(P)H-nitrate reductase from Ankistrodesmus baunii. Eur J Biochem 1 0 6 249-256 De la Rosa MA, Vega JM, Zumft WG (1981) Composition and structure of assimilatory nitrate reductase from Ankistrodesmus baunii. J Biol Chem 256 5814-5819 Deng M-D, Moureaux T, Cherel I, Boutin J-I', Caboche M (1991) Effects of nitrogen metabolites on the regulation and circadian expression of tobacco nitrate reductase. Plant Physiol Biochem 2 9 239-247 Deng M-D, Moureaux T, Leydecker M-T, Caboche M (1990) Nitrate-reductase expression is under the control of a circadian rhythm and is light inducible in Nicotiana tabacum leaves. Planta 1 8 0 257-261
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Dunlap JC, Hastings JW (1981) The biological clock m Gonyaulax controls luciferase activity by regulating turnover. J Biol Chem 256 10509-10518 Fiddo RJ, Notton BA (1984) Spinach nitrate reductase: further purification
