Nitrate reductase from squash: cDNA cloning and ... - Europe PMC

Proc. Natl. Acad. Sci. USA Vol. 83, pp. 8073-8076, November 1986 Biochemistry

Nitrate reductase from squash: cDNA cloning and nitrate regulation (plant gene expression/Xgtl1 cDNA cloning)

NIGEL M. CRAWFORD*, WILBUR H. CAMPBELLt, AND RONALD W. DAVIS* *Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305; and tDepartment of Biological Sciences, Michigan Technological University, Houghton, MI 49931

Contributed by Ronald W. Davis, July 31, 1986

The assimilation of nitrate in plants involves ABSTRACT the reduction of nitrate to ammonla in two steps. The first step requires nitrate reductase, a nitrate-inducible enzyme. When seedlings of squash (Cucurbita maxima L.) were treated with nitrate, both nitrate reductase activity and protein were induced in the cotyledons. Poly(A)+ RNA was prepared from cotyledons of nitrate-treated seedlings and was used to construct a Agtll cDNA library. Using antibodies from mice immunized against purified nitrate reductase from squash, a recombinant A phage was isolated that encoded part of the nitrate reductase mRNA. The antigens produced by the recombinant phage were used to affnity purify anti-nitrate reductase antibody from ascites fluid of immunized mice. The purified antibody bound to nitrate reductase protein on immunoblots and immunoprecipitated the enzyme from squash protein extracts. The cDNA insert (1.2 kilobases) hybridized to a 3.2-kilobase RNA that was 120-fold more abundant in nitrateinduced cotyledons compared with the uninduced tissue. Most higher plants acquire the bulk of their nitrogen in the form of nitrate in the soil (for reviews, see refs. 1-5). Nitrate is efficiently taken up by the roots and then reduced to ammonia in two steps: the first step requires the enzyme nitrate reductase (NR); the second requires nitrite reductase (NiR): NR

NO3

NiR

NO2 - NH+

In many plants, including squash, most of the nitrate is reduced in the leaves (2, 5). In these plants, the nitrate taken up by the roots is transported through the xylem to the shoot

(5, 6).

The key regulatory step in the nitrate assimilation pathway is the first reaction, the reduction of nitrate. The nitrate reductase enzyme is the rate-limiting factor for the conversion of nitrate to ammonia and is highly regulated (1-5). Factors such as light (7-11) and the phytohormone cytokinin (12) increase the levels of the enzyme. Most important, nitrate reductase is induced by its substrate nitrate (7, 10, 11, 13, 14). The levels of both nitrate reductase activity and protein increase dramatically when plants or tissue explants are treated with nitrate. Maximal levels are usually attained within 24-36 hr after a single application of nitrate. The molecular mechanism for the induction of the enzyme is not known. Nitrate reductase has been purified and characterized from many different plants (reviewed in ref. 2). The assay for nitrate reduction is rapid, and the enzyme can be purified several hundred-fold in a single chromatographic step (15). It was purified to homogeneity from squash by affinity chromatography and was found to be a homodimer of 115-kDa The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

subunits (16, 17). Each subunit of the enzyme contains 1 equivalent of FAD, heme iron, and molybdenum-pterin as prosthetic groups (17). Antibodies to squash nitrate reductase have been prepared and used for immunological studies of the enzyme (18). We use these antibodies to characterize the nitrate induction of the enzyme in squash and to isolate a cDNA clone encoding the enzyme using the X expression vector Xgtll (19). Our goal is to test whether the level of nitrate reductase mRNA can be regulated by nitrate.

MATERIALS AND METHODS Plant Material. Squash (Cucurbita maxima L. cv Buttercup) was grown in vermiculite as described (15). Three days after planting, plants were watered with a modified Hoagland's solution containing 35 mM NH4NO3 (for induction) or 35 mM NH4Cl (for control). One day later the seedlings were again watered with nutrient medium. Seventeen hours after the second watering, the cotyledons were harvested and frozen in liquid N2. Protein Procedures. Frozen cotyledons were powdered with a mortar and pestle, then resuspended into 100 mM KPO4, pH 7.5/1 mM Na2EDTA/2 mM Na2EGTA, using 5 ml of buffer per g (fresh weight) of tissue. The extract was centrifuged at 10,000 x g for 10 min. The supernatant (S10) was stored at -70°C. For affinity chromatography, 15 ml of the S10 was chromatographed as described (16) except that Affi-Gel blue agarose (Bio-Rad) was used. Nitrate reductase was purified 220-fold by this step with a 30% yield. Nitrate reductase assays were performed as described (16); 1 unit of activity is defined as the production of 1 ,umol of nitrite per min at 30°C. Mouse antibodies (ascites fluid) against gel-purified nitrate reductase from squash were prepared by Jill Remmler (State University of New York, Syracuse) (20). Control ascites fluid was prepared in the same manner, except that mice were injected with polymerized acrylamide. Immunoblot analysis (21) was performed as described (20), except for the following changes. Nitrocellulose filters with bound proteins were incubated with 50 mM Tris-HCl, pH 8.0/150 mM NaCl/ 0.05% Tween 20 (wash buffer) with 20% fetal calf serum for 30 min at 20°C. Filters were then incubated for 60 min at 20°C with mouse ascites fluid diluted 1:250 in wash buffer, washed three times for 5 min each at 20°C with wash buffer, incubated with "25I-labeled protein A (45 uCi/,g; 1 Ci = 37 GBq; ICN) at 0.1 ,tCi/ml for 30 min at 20°C in wash buffer, then washed four times for 5 min each at 20°C with wash buffer. Filters were dried and then autoradiographed at -70°C. To affimitypurify antibody, extracts of bacteria infected with recombinant X phage were immobilized onto nitrocellulose filters and then bound to antibodies in the immune ascites fluid as described (22). A filter (3 cm2) was used for 50 Al of ascites fluid in 0.5 ml of wash buffer. Bound antibody was eluted Abbreviation: kb, kilobase(s).

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from the filters with 0.5 ml of 200 mM glycine hydrochloride (pH 2.5), neutralized with 62 ,ul of 1 M Tris base, and then either added directly to the squash extract (S10) to immunoprecipitate protein with protein A-Sepharose (Sigma) or diluted 1:1 with 200 mM NaCl/0.1% Tween 20, and incubated with the immunoblots. Nucleic Acid Procedures. Poly(A)+ RNA was isolated from frozen squash cotyledons as described (23), except that 25 mM dithiothreitol was added to the extraction buffer, and the RNA was used directly after oligo(dT)-cellulose chromatography. A yield of 4 ,g of poly(A)+ RNA per g (fresh weight) of tissue was obtained. RNA blot analysis (24) was performed by transferring glyoxal-treated RNA separated by agarose gel electrophoresis (25) to positively charged nylon membranes (Genatran 45, Plasco, Woburn, MA). Hybridizations were done in 50% formamide/4x SSPE (26)/5x BFP (26)/0.2 mg of DNA per ml/1% NaDodSO4/5% sodium Dextran sulfate/0.2 mg of poly(U) per ml at 42°C. Low-stringency hybridizations were in 30% formamide/4 x SSPE/5 x BFP/0.2 mg of DNA per ml/1% NaDodSO4/5% sodium Dextran sulfate/0.2 mg of poly(U) per ml at 37°C. DNA was radiolabeled to >1 x 10 cpm/,ug as described (27) and radiolabeled DNA (1 x 106 cpm/ml) was used in the hybridizations. The cDNA clone for carrot a-tubulin was a gift from Renee Sung (University of California, Berkeley). cDNA Library. cDNA was synthesized from poly(A)+ RNA prepared as described (28, 29) from nitrate-treated cotyledons, except for the following changes. First-strand DNA was synthesized by incubating 7.5 ,g of RNA at 42°C for 120 min in 50 mM Tris HCl, pH 8.5/40 mM KCl/6 mM MgCl2/0.4 mM dithiothreitol/40 ,ug of oligo(dT) per ml/200 ,uCi of [a-32P]dCTP per ml/1.5 mM each dATP, dGTP, dTTP/0.5 mM dCTP/1000 units of reverse transcriptase per ml. Second-strand DNA was synthesized at .15C for 2 hr in 100 mM Hepes, pH 6.9/70 mM KCl/6 mM MgCl2/15 mM 2-mercaptoethanol/0.1 mM each dATP, dGTP, dCTP, dTTP/12 units of RNase H per ml/400 units of DNA polymerase I per ml. The cDNA was then prepared for ligation with Xgtll DNA (Promega Biotec, Madison, WI) and packaged into phage particles. Of the total number of phage, 80% were recombinant and 3 x 106 recombinant phage were obtained per ,ug of poly(A)+ RNA. The unamplified Xgtll cDNA library was screened as described (22).

Table 1. Nitrate reductase activity in squash extracts and its inhibition by anti-NR antibodies % Nitrate induction Activity, inhibition of tissue units x 10-3 Antibody U 200fold by affinity chromatography (lane 6). About 5-10%o of the protein in this enriched fraction should be nitrate reductase (16). We conclude that the level of nitrate reductase protein kDa 200 -

RESULTS Induction of Nitrate Reductase by Nitrate. The effect of nitrate treatment of squash seedlings on nitrate reductase levels was investigated. Protein extracts were prepared from the cotyledons of seedlings 2 days after they had been irrigated with nitrate (NH4NO3)- and non-nitrate (NH4Cl)containing medium. The ammonium ion in the medium served as a convenient counterion to nitrate and does not interfere with the nitrate induction (unpublished results; ref. 11). When nitrate reductase activity was measured, activity was detected only in the extracts from nitrate-treated plants (Table 1). No inhibition of enzyme activity was found when extracts from induced and control plants were mixed prior to the assay. Next, antibody inhibition assays were performed by adding mouse ascites fluid to the protein extracts and then clearing antigen-antibody complexes from the mixture with protein A-Sepharose. Anti-nitrate reductase antibody inhibited >85% of the nitrate reductase activity in the protein extract, while control antibody (nonimmune ascites fluid) showed no inhibition (Table 1). The effect of nitrate treatment on the abundance of nitrate reductase protein was tested next. Protein in the squash extracts used for the activity assays described above was subjected to immunoblot analysis (21). Protein was separated by polyacrylamide gel electrophoresis, transferred to nitro-

(1986)

92.5 -

1

2

3

4

5

6

0_

_

4w

7

41

68-

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FIG. 1. Nitrate induction of nitrate reductase protein, immunoblot analysis. Protein extracts from cotyledons were separated by NaDodSO4/polyacrylamide gel electrophoresis (7.5% acrylamide) and then transferred to nitrocellulose filters. Filters were incubated with ascites fluid and then with 125I-labeled protein A, followed by autoradiography as described in Materials and Methods. Filters were screened with control ascites fluid (lanes 1 and 2), with immune ascites fluid (lanes 3-6), or with affinity-purified antibodies that bound to XCmcl antigens (lane 7). Onto the gel were loaded 100 ,g of protein (S10) from control tissue (lanes 1 and 3), 100 ug of protein l10) from NO--induced tissue (0.00125 unit of nitrate reductz activity) (lanes 2, 4, and 5), 0.4 ,ug of protein (0.0027 unit of nitr"*.z reductase activity) purified by Affi-Gel Blue agarose chromatography (lane 6), and 2.0 yg of the same purified protein as in lane 6 (0.0185 unit of nitrate reductase activity) (lane 7). Molecular size markers (Bethesda Research Laboratories) were prestained.

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in the cotyledons is increased by treating seedlings with nitrate. cDNA Cloning of Nitrate Reductase mRNA. As shown above, the antibody in the immune ascites fluid specifically recognized nitrate reductase protein. Therefore, the antibody appeared to be suitable for use as a probe in screening a cDNA expression library of squash for a cDNA clone encoding nitrate reductase. To prepare the library, total RNA was prepared from cotyledons of nitrate-treated seedlings by phenol extraction. Poly(A)+ RNA was purified by oligo(dT)cellulose chromatography. Double-stranded cDNA between 1.0 and 4.0 kilobases (kb) was prepared, ligated to Xgtll vector DNA, and then packaged into phage particles. Recombinant phage (250,000) were screened with the anti-nitrate reductase antibody, and one positive phage (XCmcl) was detected and purified. To verify that antibodies in the immune ascites fluid were specifically recognizing protein encoded by XCmcl, plaquepurified phage were immunoscreened with ascites fluid (Fig. 2). No specific binding of antibody was detected to XCmcl antigens with control ascites fluid (Fig. 2A) or to antigens produced by the parental phage Xgtl1 with immune ascites fluid (Fig. 2B). As expected, strong binding ofimmune ascites fluid to the XCmcl antigens was observed (Fig. 2C). Thus, antibody present only in the immune ascites fluid bound to the specific antigens produced by the recombinant phage xCmcl.

A

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To demonstrate that XCmcl contained a cDNA encoding at least part of the nitrate reductase mRNA, the proteins encoded by ACmcl were used to affinity-purify antibody from ascites fluid. Plate lysates from bacteria infected with XCmcl were transferred to nitrocellulose, and the bound protein was used to purify reactive antibody. After the filter was washed, bound antibody was eluted and tested. The affinity-purified antibody from immune ascites fluid inhibited most of the nitrate reductase activity, clearing almost 90% of the nitrate reductase protein from the extract (Table 1). The clearing step was not necessary for the inhibition; if the protein A-Sepharose was omitted, the same level of inhibition was observed. The purified antibody from control ascites fluid had no effect on the enzymatic activity (Table 1). The purified anti-nitrate reductase antibody was also tested by immunoblot analysis. The partially purified nitrate reductase preparation was transferred to nitrocellulose after NaDodSO4/polyacrylamide gel electrophoresis. The affinitypurified antibody specifically recognized a protein with the expected molecular mass for nitrate reductase (Fig. 1, lane 7). Therefore, the cDNA insert of XCmcl encodes protein with antigenic determinants identical to nitrate reductase. RNA Analysis. The effect of nitrate on the level of nitrate reductase mRNA could now be tested. First, the cDNA insert of XCmcl was gel-purified and found to be 1.2 kb long. Poly(A)+ RNA was prepared from the same batch of cotyledons used for the protein and activity assays described above. The RNA was resolved by agarose gel electrophoresis, transferred to nitrocellulose filters, and hybridized to radiolabeled cDNA (Fig. 3A). A band was detected corresponding to a 3.2-kb RNA found only in nitrate-treated tissue (lane 2). No hybridization was seen to RNA from control 'tissue (lane 1). With a much longer exposure, a faint band with