Biochimica et BiophysicaActg 1088 (1991) 127-130 © 1991 ElsevierSciencePublishersB.V.(BiomedicalDivision)0167-4781/91/$03.50 ADONIS 016747819100066F

BBAEXP 90202

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Regulation of the inducible nitrate reductase isoform from Soybeans J o h n J. C a l l a c i a n d J o h n S m a r r e l l i , Jr. Department of Biology, Loyola Universityof Chicago, Chicago, IL (U.S.A.)

(Received13 August 1990) Key words: Nitrate reductase;Transcriptionrate; Nitrate induction The activity of the pH 7.5 NADH-linked nitrate reductase isoform from soybeans is termed inducible. Activity can be observed only in seedlings which have been supplied nitrate. Steady-state levels of mRNA for this is~orm also show an absolute requirement for nitrate. Nitrate reductase specific mRNA can be observed within 2 h after nitrate treatment. Levels peaked 48 h after nitrate treatment, while the addition of glutamine to nitrate diminished amounts of nitrate reductase specific mRNA. Using nuclear runoff transcription assays, we have shown that one level of control of nitrate reductase synthesis is transcriptional. The majority of inorganic nitrogen converted into organic nitrogen by higher plants is derived from the assimilation of nitrate [1]: nitrate 2e- ~nitrate 6e-~ammonium_~glutamate These nitrate reduction reactions occur predominantly in the leaves of most plants [1]. The first step in this pathway, catalyzed by nitrate reductase, may be considered rate-limiting and thus is highly regulated [1]. Factors such as light and nitrogen availability have been shown to be very important in modulating nitrate reductase activity [3,4]. Nitrate reductases are soluble electron-transferring proteins [1]. In most higher plants, the enzyme is a homodimer of 110-115 kDa subunits, each containing flavin (FAD), cytochrome b-557 and molybdenum comprising three redox centers [1]. These groups transfer electrons between the pyridine nucleotide oxidation site and the nitrate reduction site [1]. Soybean leaves have been shown to contain three isoforms of nitrate reductase [5]. Two of the isoforms have been termed constitutive, as their activities can be observed in seedlings not provided with nitrate [6]. However, their activities do increase in nitrate treated plants [6]. The third isoform is termed inducible; the treatment of seedlings with nitrate is a prerequisite for its expression and activity [2]. This study focused on the molecular events which

Correspondence: J. Smarrelli, Jr., Department of Biology, Loyola University of Chicago, 6525 N. Sheridan Rd., Chicago, IL 60626, U.S.A.

regulate expression of the inducible nitrate reductase isoform in soybeans. The overall objective was to determine if expression of the inducible nitrate reductase isoform was controlled at the level of transcription. A 1.2 kb eDNA clone for squash nitrate reductase [7] was used as a probe for inducible nitrate reductase mRNA. This clone was utilized to quantitate RNA levels for both the steady-state mRNA experiments and nuclear runoff transcription assays. Soybean seedlings (Glycine max vat. Williams 82) were grown in vermiculite and treated as previously described [6]. Starting on day 10 ~fter planting and 4 h after the beginning of the photoperiod, the vermiculite was saturated with Hoagland's nutrient solution [6] providing the following as sole nitrogen sources: (1) no nitrogen; (2) 50 mM nitrate; (3) 10 mM ghitamine; (4) 50 mM nitrate and 10 mM glutamine. Random samples of primary leaves were harvested, frozen in liquid nitrogen and stored at - 8 0 ° C prior to RNA isolations. Total cellular R N A was isolated as previously described [8]. After ethanol precipitation, poly(A) + mRNA was purified using oligo(dT)-cellulose chromatography as described [8]. Samples were precipitated with ethanol, dissolved in H20 and stored at - 8 0 ° C . Approx. 30 pg of poly(A) + m R N A was obtained from each 10 g sample, with an A~o/A28o ratio of at least 1.8. Electrophoresis of glyoxal and dimethylsulfoxide denatured RNA samples was performed in 1~ agarose as described [9]. Slot blots were performed on glyoxal denatured R N A samples as described by Thomas [10]. RNA samples were hybridized with plasmid pCmcl obt~ned from Dr. Nigel Crawford. This plasmid has a 1.2 kb cDNA insert for squash nitrate reductase, and lay-

128 bridizes with an approx. 3 kb mRNA from nitrate induced squash [7]. [3~p]dCTP was incorporated into the plasmid using a nick translation kit obtained from BRL. Unincorporated label was selaarated from labelled DNA using Sephadex G-50. Nuclei from 5-day-old soybean leaf tissues were isolated by a modification of the method described by Hagen and Guilfoyle [11]. All steps were performed at 2 ° to 4°C. Nucleus isolation buffer (10 mM Tris-HC1 (pH 7.2), 5 mM MgCl 2, 10 mM 2-mercaptoethanol and 1 M sucrose) was added to 5 g of tissue at 10 vol. per g fresh tissue weight and homogenized for 45 s with a Brinkman Polytron. The homogenate was filtered through four layers of cheesecloth and then through 300, 100 and 50 am mesh. 25% Triton was added to the filtrate to achieve a final concentration of 1%, and the suspension filtered through a 20 am filter. The filtrate was then centrifuged at 5000 rpm for 10 rain in a HS-4 swinging bucket rotor and the pellet was resuspended in 5 rnl nucleus isolation buffer. The suspension was then layered over a discontinuous Percoll gradient containing 5 ml 50% Percoll and 5 ml 25% Percoll in nucleus isolation buffer. The gradient was centrifuged at 5000 rpm for 30 rain in a HS.4 rotor. Nuclei banded at the interface between the 25% and the 50% Percoll layers, and were removed with a Pasteur pipet. The nuclei were pelleted twice in nucleus isolation buffer to remove residual Percoll. Purity of isolated nuclei was monitored by light microscopy. The nuclei were resuspended at a concentration of I • 10 s per ml in nuclear storage buffer (20 mM Hews (pH 7.2), 5 mM MgC! 2, 2 mM dithiothrcitol (DTT) and 50% glycerol) and stored at - 80 ° C. Transcriptional activity of isolated nuclei was measured by the total incorporation of [3H]UTP in an in vitro transcription assay [11]. 80 units of RNasin (Promega Biotech, Madison, WI) was added to 106 soybean nuclei and incubated at 27°C. The nuclei were then added to a solution at 27 °C containing 20 mM Hepes (pH 7.9), 12.5 mM MgCI 2, 100 mM (NH4)2 SO4, 1.0 mM DTT, 25% (v/v) glycerol, 0.5 mM each A T E CTP and GTP, and 100 /iCi of [3H]UTP (New England Nuclear, Boston, MA). Aliquots of the reaction mixture were removed at time zero and every 5 rain thereafter throughout a 25 min period. The aliquots were spotted ontc, Whatman glass filters (GFC) pre-wetted with icecold 10% trichloroacetic acid (TCA). The filters were washed three times each with ice-cold 10% TCA and dried with 95% ethanol. Labelled RNA bound to the filters was detected using liquid scintillation counting. Duplicate filters were counted for each time point and average epm were recorded. Nuclei shown to be transcriptionally active by incorporation assays were then used for nuclear runoff transcription assays, which measured levels of inducible nitrate reductase mRNA transcribed in vitro. These assays were performed as described by Hagen and

Guilfoyle [11]. 5- 106 nuclei were incubated in 200/tl of a solution containing 20 mM Hepes (pH 7.9), 12.5 mM MgCI 2, 100 mM (NH4):SO 4, 1.0 mM DTT, 25% (v/v) glycerol, 0.5 mM each ATP, CTP and GTP, 100/LCi of [32p]UTP (600 Ci/mmol) and 160 units RNasir, Assays were conducted at 30°C for 10 rain. After 10 rain, nuclei were treated with 12 /Lg of DNase I (Sigma Chemical, St. Louis, MO) for 5 rain at 25°C. Next, a 400 #1 vol. of 7.5 M urea, 5% SDS, 20 mM EDTA, 100 mM LiCI and 10 mM aurintricarboxylic acid (ATA) (pH 7.0) was added along with 600 /L! of phenol/ chioroform/isoamyl alcohol (25:24:1), and the solution was vortexed for 1 rain. The mixture was then centrifuged for 15 rain at 4 ° C in a microfuge. The aqueous phase was collected and to it was added 200/tl of 4 M ammonium acetate, 20/~g wheat-germ tRNA, and 2.5 vol. of 100~ ethanol. The mixture was then incubated at - 2 0 ° C for 12 h. RNA was pelleted at 4 ° C in a microfuge. The pellet was washed with 70% ethanol, dried and resuspended in 10 ~tl sterile H20. To quantify levels of inducible nitrate reductase transcript produced in the nuclear runoff assays, hybridizations were carried out in DNA excess, as described [11]. 5/tg amounts of EcoRl-digested pCmcl were used for these hybridizations. Poly(A) + mRNA was isolated from primary leaves of 10-day-old soybean seedlings supplied with various nitrogen regimes. All samples isolated had A26o/A2so ratios above 1.8, and no differences could be detected among RNAs isolated from seedlings provided with different nitrogen sources as evidenced by agarose gel electrophoresis. Northern blots using the 1.2 kb squash cDNA nitrate reductase clone as a probe revealed a single band of 3.3 kb, found only with RNA isolated from plants supplied nitrate (data not shown). Equal quantities of poly(A) + RNA isolated from leaves harvested at various times after nutrient treatment were subsequently probed for the presence of inducible nitrate reductase transcripts. The results obtained from densitometric quantitations of slot blots are shown in Table I. No hybridization was observed using RNA from plants not provided nitrate. Maximal steady-state mRNA levels for plants treated with nitrate alone or with nitrate and glutamine were observed 48 h after treatment. Nitrate inducible transcripts could be observed within 2 h after treatment. Further, in the dark, the levels of nitrate reductase specific mRNA dramatically decreased. Transcriptional activity of the nitrate reductase gene was subsequently assessed by performing nuclear runoff transcription assays. Nuclei were isolated from 5-day-old soybean seedlings using Percoll gradients. The purest nuclear preparations (approx. l0 s nuclei per 5 g tissue) were obtained from the leaves of 5-day-old seedlings. Since the steady-state mRNA results were identical for both 5- and 10-day-old seedlings (data not shown),

129 TABLE !

Densitometric quontitation of slot blots. Time-course for metabolite control of steady-state messenger RNA levels for the inducible nitrate reductase isoform from soybeans

~T ' ~

5 lag of glyoxal-treated poly(A) + RNA isolated from the leaves of soybean plants treated as shown were slotted onto Genescreen and hybridized with the 32P-labelled squash c D N A probe. After washing and autoradi~graphy, densitometry was used to quantitate the hybridization signal Time (h)

0

2 4 10 24 42 (dark) 48

10

O0

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4

Peak height (cm) Nitrate

Nitrate + Glutamine

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0.9 0.9 2.2 1.3 2.3

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10

15

Time (minutes)

20

25

30

Nittote/GlutomineTreoted Nuclei

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nuclei were isolated from younger seedlings. Nuclear runoff transcription assays were performed with equal numbers of nuclei isolated at the times indicated, from plants given various nutrient treatments (Fig. 1A, B). Linear incorporation of label is observed for nuclei isolated from all samples, as nuclei isolated from plants in the dark period showed the lowest transcriptional activity. Subsequently, labelled transcripts were used to probe the squash clone at DNA excess. Quantitation of nitrate reductase gene transcriptional activity is shown in Table II. At time zero, no signal could be observed. An increased level of hybridization was observed from assays using nuclei isolated 4, 24 and 48 h after nitrate treatment. Nuclei isolated from plants, which had entered the dark period (42 h), showed no hybridization. Experiments performed in this study quantitated steady-state mRNA levels for the inducible nitrate reductase isoform from soybeans, as well as measuring transcriptional activity of the gene for this isoform in response to nitrogen metabolites. The goal of these studies was to determine whether the synthesis of substrate-inducible nitrate reductase was controlled at the level of transcription. Using a cDNA clone that encodes part of the mRNA for nitrate reductase from squash as a specific probe to the pH 7.5 NADH-linked nitrate reductase from soybeans, it has been shown that soybean seedlings not supplied with nitrate possess no measurable amounts of transcript for the inducible nitrate reductase isoform. These results confirm previous enzymological studies [6], and are consistent with the role of nitrate in the appearance of nitrate reductase mRNA from soybeans [2], corn [12], barley [13,18], Arabidopsis [14], tomato and tobacco [15], and squash [7]. Further, we have described glutamine inhibition of levels of steady-state

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Fig. t. Transcriptional competence of isolated nuclei. Incorporation of 13H]UTP into TCA-precipitable counts was measured for nuclei isolated at various time points after treatment. Each point is the average

of duplicate values using 106 nuclei and 100 ~Ci of [3HIUTP in a standard reaction mixture. Lines are drawn by linear regression.

mRNA. Although glutamine repression of nitrate reductase synthesis is well documented in fungi [16], the role of glutamine and other amino acids in the regulation of nitrate reductase remains controversial [17]. Our results have demonstrated that the gene for the TABLE 11

Densitometer measurements of nuclear runoff transcription assay hybridizations. Transcriptional control of the reducible nitrate reductase isoform from soybeans 5/t 8 of the squash c D N A clone was digested with EcoRl, slotted onto Genescreen, and hybridized to the 132p]CTP labelled transcripts (approx. 10 6 clam from each treatment) from 5.10 6 nuclei isolated at the times indicated after nitrate treatment. Blots were exposed to X-ray film for 5 days. Densitometry was used to quantitative peak heights Time after nitrate (h)

Peak height (t.~)

0 4 24 42 (dark)

0.2 2.75 3.25 0.3

48

5.3

130 i n d u c i b l e n i t r a t e r e d u c t a s e i s o f o r m is n o t t r a n s c r i b e d u n l e s s n i t r a t e is a v a i l a b l e to t h e plant. W e a r e c u r r e n t l y p u r s u i n g cis- a n d trans-acting r e g u l a t o r y e l e m e n t s to f u r t h e r delineate t h e m o l e c u l a r c o n t r o l 6 f t h e s y n t h e s i s of the inducible nitrate reductase isoform. T h e s u p p o r t o f the N a t i o n a l Science F o u n d a l i o n ( D M B 87-07243) to J.S. is g r a t e f u l l y a c k n o w l e d g e d , In addition, this w o r k w a s s u p p o r t e d b y a S u m m e r Research A w a r d f r o m L o y o l a U n i v e r s i t y to J.S.

References 1 Campbell, W.H. and Smarrelli, J. (1986) in Biochemical Basis of Plant Breeding (Neyra, C.A., ed.). Vol 2, pp. 1-39, CRC Press, Boca Raton, FL. 2 SmarreUi, J.. Jr., Malone, M.J., Watters, M.T. and Curtis, L.T. (1986) Biochem. Biophys. Res. Commun. 146, 1160-1165. 3 Gupta, S.C. and Beevers, L. (1983) J. Exp. Botany 34, 1455-1462. 4 Somers, D.A.. Kuo, T.M., Kleinhofs, A., Warner, R.L. and Oaks, A. (1983) 7. Plant Physiol. 72. 949-952. 5 Streit, L., Nelson, R.S. and Harper, J.E. (1985) J. Plant Physiol. 78, 80-84.

6 Curtis. L.T. and Smarrelli, J., Jr. (1986) J. Plant Physiol. 126. 135-143. 7 Crawford, N.. Campbell, W.H. and Davis, R. (1986) Proc. Natl. Acad. Sci. USA 83, 8073-8076. 8 *lartino, S.J. and Smarrelli, J.. Jr. (1989) Plant Sci. 61, 61-67. 9 Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York. 10 Thomas, P.S. (1983) Methods Enzymol. 100. 255-266. 11 Hagen, G. and Guilfoyle, T.J. (1985) Mol. Cell Biol. 5. 1197-1203. 12 Gowri, (3. and Campbell, W.H. (1989) J. Plant Physiol. 90, 792798. 13 Cheng, C.L., Dewdney, J., Kleinhofs, A. and Goodr01an, H.M. (1986) Proc. Natl. Acad. Sci. USA 83, 6825-6828. 14 Crawford, N.M., Smith, M., Bellissimo. D. and Davis, R. (1988) proc. Natl. Acad. Sci. USA 85, 5006-5010. 15 Galangau, F., DanieI-Vedele, ::., Moureaux, T., Dorbe, M.F., Leydecker. M.T. and Caboche, M. (1988) J. Plant Physiol. 88, 383-388. 16 Fu, Y.H. and Marzluf, G.A. (1987) proc. Natl. Acad. Sci. USA 84, 8243-8247. 17 Caboche, M. and Rouze, P. (1990) Trends Genet. 6, 187-192. 18 Lu, J., Ertl, J.R. and Chen, C. (1990) Plant Mol. Biol. 14, 585-594.

Regulation of the inducible nitrate reductase isoform from soybeans.

The activity of the pH 7.5 NADH-linked nitrate reductase isoform from soybeans is termed inducible. Activity can be observed only in seedlings which h...
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