AU639026B2 - A membrane associated protein from plants which has nitrate reductase activity - Google Patents

Description

WO 91/04325 PCT/US90/04397 I TITLE OF THE INVENTION: A membrane associated protein from plants which has nitrate reductase activity.

Field of the Invention: The invention pertains to a nitrate reductase activity of plants, and in particular of barley seedlings. The invention further pertains to recombinant molecules which express this activity, to transformed plants which express such activity, and to the uses of this activity in agriculture.

This invention was made in part with support from grant NCC2-99 of the National Aeronautics and Space Administration (NASA). The government has attained rights in this invention.

BACKGROUND OF THE INVENTION Nitrate (N0 3 is the major source of nitrogen for higher plants. Such nitrate must be transported into the plant's cells, and then chemically reduced to ammonia, in order to be incorporated into amino acids.

The transport of N0 3 by roots is, thus, the first step in the process of N0 3 assimilation in plants. Transport is -i WO 91/04325 PCT/US90/04397 -2induced by N03- and requires both RNA and protein synthesis, leading Jackson, W.A. et al. (Plant Physiol. 52:120-127 (1973)) to propose that a specific NO 3 transport protein is synthesized. Although much is known about the activity of the N03- transporter (Goyal, S.S. et al., Plant Phvsiol. 82:1051- 1056 (1986)), a N0 3 transport protein has not yet been identified in higher plants.

Once nitrate has been transported into the plant, it is reduced in a multi-enzyme process to ammonia. The reduction of nitrate is closely regulated since nitrate reduction requires substantial energy, and since the final product of the reduction, ammonia, is tcxic when accumulated (and must, therefore, itself be transported when present in excess).

The first enzyme in the nitrate reducing pathway is termed "Nitrate Reductase" (or, equivalently, "NR activity") (Guerrero, M.G. et al., Ann. Rev. Plant Physiol. 32:169-204 (1981)). Plant NR activity is generally thought to be localized in the cytoplasm (Ritenour, et al., Plant Physiol. 42:233-237 (1966); Dalling, et al., Biochim.

Biophys. Acta 283:513-519 (1972); Suzuki, et al., Planta 151:457.461 (1981); Oaks, A. et al., Ann. Rev. Plant Phvsiol.

36:345-365 (1985)).

Several reports have suggested that a portion of the total plant nitrate reductase activity is not cytosolic.

Miflin, for example, initially found a small percentage of the total barley root NR activity in a mitochondria enriched fraction (Miflin, Nature 214:1133-1134 (1967)).

Miflin, B.J. subsequently reported that a membrane associated NR activity was localized in an unidentified particulate fraction distinct from the mitochondria (Miflin, Planta 93:160-170 (1970)). Blevins et al. (Plant Physiol. 57:458-459 (1976)) later showed that the NR activity found in particulate fractions, prepared from barley roots, could have been due to bacterial contamination, especially when the seedlings were II WO 91/04325 PCT/US90/04397 -3grown under low 02. Lips, S.H. et al. suggested that an NR activity activity was localized on the membranes of cell microbodies in tobacco leaves and that NR activity detected in soluble fractions had been released from the membranes during tissue extraction (Lips, S.H. et al., Eur. J. Biochem. 29:20- 24 (1972)). Utilizing immunogold labeling techniques, Kamachi et al. (Plant Cell Physiol. 28(2):333-338 (1987)) recently proposed the presence of an NR activity in chloroplasts. Lopez-Ruiz et al. (Plant Phvsiol. 79:1006-1010 (1985)) localized an NR activity of green algae in the pyrenoid.

Several observations have further suggested that such a non-cytosolic NR activity may be localized in the plasma membrane of a cell. Rufty et al. (Plant Physiol.

82:675-680 (1986)), for example, discussed the possibility that the receptor for the NR activity induction system and the functional NR activity protein may both be associated with the PM of root cells. Butz, R.G. et al. (Phvtochemistry 13:409- 417 (1977)) proposed that a transmembrane NR activity may function as a carrier for N0 3 transport. Nitrate reductase activity has been found in the cell wall-PM region, and in the tonoplast membranes, of Neurospora crassa (Roldan, J.M., et al., Plant Phvsiol. 70:872-874 (1982)). Nitrate reductase activity has, however, not been found to be localized in the plasma membrane of higher plants.

Since both NH 4 and N0 3 transport are simultaneously induced by N0 3 inhibited by protein and RNA synthesis inhibitors and increased in activity by supplying glucose to roots Butz, R.G. et al. proposed that a membrane-associated NR activity functions as a carrier for NO3"transport (Butz, R.G.

et al., Phytochem. 16:409-417 (1977)).

I WO 91/04325 PCT/US90/04397 -4- SUMMARY OF THE INVENTION Latent nitrate reductase activity (NR activity) was detected in corn (Zea mays Golden Jubilee) root microsome fractions. Microsomal-associated NR activity was stimulated up to 20 fold by Triton X-100 (octyl phenoxy polyethoxyethanol) whereas soluble NR activity was only increased up to 1.2 fold. Microsomal-associated NR activity represented up to 19% of the total root NR activity.

Analysis of microsomal fractions by aqueous two-phase partitioning showed that the membrane-associated NR activity was localized in the second upper phase (U 2 Analysis with marker enzymes indicated that the U 2 fraction was plasma membrane The PM associated NR activity was not removed by washing vesicles with up to 1.0 M NaC1 but was solubilized from the PM with 0.05% Triton X-100. In contrast, vanadate sensitive ATPase activity was not solubilized from the PM by treatment with 0.1% Triton X-100. The results show that a protein capable of reducing nitrate is embedded in the hydrophobic region of the PM of corn roots.

Membrane associated nitrate reductase (NR) was detected in plasma membrane (PM) fractions isolated by aqueous twophase partitioning from barley (Hordeum vulgare L. var CM 72) roots. The PM associated NR was not removed by washing vesicles with 500 mM NaC1 and 1 mM EDTA and represented up to 4% of the total root NR activity. PM associated NR was stimulated up to 20 fold by Triton X-100 whereas soluble NR was only increased 1.7 fold. The latency was a function of the solubilization of NR from the membrane. NR, solubilized from the PM fraction by Triton X-100 was inactivated by antiserum to Chlorella sorokiniana NR. Anti-NR immunoglobulin G (IgG) fragments purified from the anti-NR serum inhibited N0 3 uptake by more than 90% but had no effect on N0 2 uptake.

The inhibitory effect was only partially reversible; uptake

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WO 91/04325 PCT/US90/04397 recovered to 50% of the control after thorough rinsing of roots. Preimmune serum IgG fragments inhibited NO 3 uptake 36% but the effect was completely reversiDle by rinsing.

Intact NR antiserum had no effect on N03- uptake. The invention shows that N03 uptake and NO 3 reduction in the PM of barley roots are related.

DESCRIPTION OF THE PREFERRED EMBODIMENTS I. Membrane Associated Nitrate Reductase The present invention concerns a "non-cytosolic," "membrane-associated" nitrate reductase activity which is derivable from higher plants. Although the invention is described in terms of the non-cytosolic, membrane-associated nitrate reductase activity of barley, it will be readily perceived that the invention permits the identification and isolation of other non-cytosolic, membrane-associated nitrate reductase activities from other plants, and that such activities, and their uses, are within the scope of the equivalents of the present invention.

The term "nitrate reductase" or as used herein, are intended to refer to enzymes capable of catalyzing the reduction of nitrate. Such enzymes may be referred to either in terms of their physical characteristics amino acid sequence, native or denatured molecular weight, etc.), or equivalently, by their functional characteristics (i.e.

kinetic constants (Vmax, Km, turnover number, etc.), substrate preferences or specificities, product(s) produced, reaction condition preferences thermal, pH, ionic, co-factor, coenzyme, etc.). The term "activity" is intended to refer to and to describe an enzyme by its functional characteristics.

An enzyme is "non-cytosolic" if it is normally not present in, or if it is substantially absent from, the _L WO 91/04325 PCT/US90/04397 -6cytoplasm of the cell in which it is naturally produced.

Thus, a non-cytosolic enzyme is one which is, or can be, present in the cytoplasm of the cell in which it is normally and naturally produced.

An enzyme is "membrane-associated" if it is bound, or capable of being naturally bound, to the plasma membrane of the cell in which it is naturally produced. Thus, a membraneassociated enzyme is one which is, or can be, present in a state bound to the plasma membrane of a cell in which it is normally and naturally produced.

II. Isolation of Plasma Membrane Fractions A highly enriched source of plasma membranes can be obtained from any of a variety of plant tissue. A preferred source of plasma membrane can be obtained from crude membrane preparations isolated from the roots of corn. Aqueous twophase partitioning (Larsson, in Modern Methods of Plant Analysis, N.S. Series, Cell Components, vol. 1, pp. 85-104, Linskens, et al., eds., Springer-Verlag, Berlin, Heidelberg, New York (1985); Hodges, and Mills, Academic Press 118:41-54 (1986); Sandstrom, et al., Plant Physiol.

85:693-698 (1987)) was used to obtain highly enriched PM fractions. A membrane-associated NR activity was localized in the PM.

Corn seedlings (Zea mays Golden Jubilee) are preferably grown hydroponically. Seed kernels are preferably treated with 1.5% volume/volume NaOCl prepared from common bleach for 15 min, rinsed with distilled water and germinated at room temperature in aerated distilled water. After approximately 24 h, the germinated seeds are spread on a stainless steel screen about 1 cm above the surface of 5 1 of an aerated 0.2 mM CaC12 solution. The seeds are covered with plastic wrapping and placed in the dark at 25'C. The plastic

L,

WO 91/04325 PCT/US90/04397 -7wrapping may be removed after 3 d, and after an additional two days, the seedlings are preferably transferred to aerated 0.1strength Hoagland solution no. 1, lacking nitrogen (Hoagland, D.R. et al., Cal. Aqric. Exp. Stat. Circ. No. 347 (1950)) and placed in a controlled environment growth chamber. The growth conditions preferably provide 60-65% relative humidity, and continuous light to minimize the effect of rhythms on NR activity levels. The photon flux density at the seedling canopy is approximately 400 gmol.m-2.s- as measured at 400- 700 nm with a Licor 190 S sensor and is preferably supplied by incandescent and cool white fluorescent lamps, respectively, at a ratio of 1:5. Lights are preferably obtained from Sylvania Lighting System, Danvers, MA. After 2 d in continuous light the seedlings may be transferred to 0.1 strength Hoagland solution no. 1 containing 1 mM KNO 3 for 24 h to induce NR activity.

The roots (approximately 25 g) from 9-d-old corn seedlings are excised, preferably just below the seed, and snipped into 1 cm pieces with a pair of scissors. The seedlings are then ground in a suitable amount of cold grinding buffer 60 ml) using a chilled mortar and pestle, or other suitable means. A suitable grinding buffer preferably comprises 250 mM sucrose, 3 mM ethylenediaminetetraacetic acid (EDTA), 50 mM 2-amino-2-(hydroxymethyl)-1,3propanediol (Tris-HC1) (pH 0.05% (w/w roots) polyvinylpolypyrrolidone, 1 mM dithiothreitol (DTT), 3 mM phenylmethylsulfonyl fluoride (PMSF), 1 uM Na 2 MoO 4

(H

2 0) 2 and tiM flavine-adenine dinucleotide (FAD). The PMSF is preferably made up as a 0.1 M stock in isopropanol and added at a ratio of 0.75 ml PMSF: 25 ml grind buffer. The DTT is preferably made up as a 1.0 M stock in deionized water and added at a ratio of 0.025 ml DTT: 25 ml grind buffer. All chemicals used in the grind buffer may be obtained from the Sigma Chemical Co., St. Louis, MO, USA. The homogenate is WO 91/04325 PCT/US90/04397 -8preferably filtered through 4 layers of cheesecloth and centrifuged preferably at 12,000 g for 10 min. The post- 12,000 g fraction may be saved for enzyme analysis. The supernatant is preferably centrifuged at 120,000 g for 20 min.

The resulting pellet represented a microsomal fraction, i.e., a crude membrane fraction. The post-120,000 g fraction may also be used for enzyme analysis.

For determinations of NR activity, the above-obtained microsomal pellets are preferably suspended in resuspension buffer, diluted to 20 ml, and centrifuged at 120,000 g for min. The resuspension buffer preferably contains 2 mM Tris- HC1 (pH 1 (M Na 2 MoO 4

(H

2 0) 2 5 IM FAD, 3 mM PMSF and 1 mM DTT. The process is preferably repeated three more times, and samples from each membrane pellet including the initial microsome fraction are saved to determine NR activity.

Microsomal pellets may alternatively be preferably resuspended in 2.2 ml of 250 mM sucrose, 5 mM K-phosphate (pH 7.8) and 1 mM PMSF, and partitioned in an aqueous polymer twophase system as detailed by Larsson et al. (Plasma Membranes, In: Modern Methods of Plant Analysis, New Series, Vol. 1, Cell Components, HF Linksens, et al., eds., Springer-Verlag, Berlin, pp. 85-104 (1985)). All steps in the procedure are preferably carried out at 4'C.

A suitable amount of the resuspended microsome fraction (1-5 ml) is preferably added to an approximately 8 ml twophase system that contained 6.5% Dextran T 500 (Pharmacia Fine Chemicals, Piscataway, NJ, USA), 6.5% (w/w) polyethylene glycol 3350 (Sigma Chemical Co., St. Louis, MO, USA), 0.33 M sucrose, 3 mM KC1 and 5 mM K-phosphate (pH 7.8).

The contents of the tubes is mixed by inversion 40-50 times, and centrifuged in a swinging-bucket rotor at 1,200 g for min.

The upper phase is collected and repartitioned on a fresh lower phase as described by Sandstrom et al. (Plant Physiol.

WO 91/04325 PCT/US90/04397 -9- 85:693-698 (1987)). The remaining microsome (U 2 and the first lower phase (L 1 fractions are collected and each is diluted with a suitable amount (such as 20 ml) of resuspension buffer and centrifuged at 120,000 g for 20 min. The U 2 pellet is suspended in resuspension buffer, diluted to 20 ml and again centrifuged at 120,000 g for 20 min. The resulting pellets are suspended and diluted to a protein concentration equivalent to the post-120,000 g fraction in resuspension buffer, and may be used directly for enzyme analysis.

Twice-centrifuged U 2 fractions are diluted to 0.5 mg.ml_ 1 protein concentration in resuspension buffer and brought to a final concentration of 0-1.0 M NaCl in 0.5 ml. All fractions are vigorously mixed for 30 sec and placed on ice. The salt treated fractions were sonicated on ice in, for example, an American Brand sonicator (American Scientific Product, McGraw Park, IL, USA). After 20 min, the fractions are diluted with 8 ml of resuspension buffer and centrifuged at 150,000 g for min. Nitrate reductase activities may be determined in the resuspended pellets.

Twice-pelleted U 2 fractions may, alternatively, be diluted to 0.5 mg/ml protein with resuspension buffer, and brought to 0 to 0.1% Triton X-100 (octyl phenoxy polyethoxyethanol, Sigma Chemical Co., St. Louis, MO, USA) in a total volume of 0.5 ml. The material is then preferably mixed vigorously for approximately 30 s and placed on ice.

After 15 min, fractions are centrifuged at 150,000 g for min without dilution. Vanadate-sensitive ATPase activity and NR activity were determined in the resuspended pellets and the soluble fractions.

The activities of various enzymes whose use facilitate the present invention may be determined in the following manner. Triton X-100-stimulated nucleoside diphosphatase (UDPase) activity is preferably assayed according to Nagahashi, J. et al. (Protoplasm 112:167-173 (1982)). Triose WO 91/04325 PCT/US90/04397 phosphate isomerase activity is preferably determined according to the method of Gibbs, M. et al., (Enzymes of glycolysis, In HF Linksens, BD Sanwal, M Tracey, eds., Modern Methods of Plant Analysis, Vol. 7, Springer Verlag Berlin, p.

520, (1964). Cytochrome c oxidase (EC is preferably assayed according to the method of Hodges, et al.

(Methods Enzymol 12:392-406 (1974)). Antimycin A insensitive NADH Cyt c reductase (EC 1.6.99.3) activity is preferably assayed as described in Lord et al. Cell Biol. 57:659-667 (1973)) and Hodges, T.K. et al. (Methods Enzymol 32:392-406 (1974)). Vanadate sensitive ATPase (EC 3.6.1.3) activity is determined in the presence and absence of Triton X-100 (Gallagher, et al., Plant Physiol. 70:1335-1340 (1982); Sandstrom, et al., Plant Phvsiol. 85:693-698 (1987)).

Nitrate sensitive ATPase activity is preferably determined according to O'Neill et al. (Plant Physiol. 72:837-846 (1983)). Alcohol dehydrogenase (EC 1.1.1.1) is preferably assayed according to the method of Suzuki, Y. et al. (Physiol.

Plant 27:121-125 (1972)). Nitrate reductase activity (EC 1.6.6.1) is preferably determined according to the method of Aslam et al. (Plant Physiol. 83:579-584 (1987)) in the presence of various concentrations of Triton X-100 (at a known T:p ratio) except that the assay volume was reduced to 500 pl.

All enzyme specific activities are reported on a mg protein basis.

Protein concentration is preferably measured using a modification of the Bradford (Bradford, Anal. Biochem.

72:248-254) method in which 60 Al of 0.2% Triton X-100 was included in each assay tube to solubilize membrane proteins; crystalline bovine serum albumin (Sigma Chemical Co., St.

Louis, MO, USA) was used as a standard.

Using the above-described methods nitrate reductase activity was detected in a crude membrane fraction isolated from corn roots. To release potentially trapped soluble NR WO 91/04325 PCT/US90/04397 -11activity, the membrane pellet may be washed, preferably four times, in hypotonic buffer (Pupillo, P. et al., Planta 151:506-511 (1981)). The initial wash releases approximately 20-25% of the NR activity to the soluble fraction. This probably represents soluble NR activity that was loosely bound or trapped within membrane vesicles. Subsequent washes of the membrane pellet release little additional NR activity. This indicates that NR activity detected in the washed membrane pellets was membrane-associated. In general, the membrane fractions are preferably washed at least two times with hypotonic buffer to remove loosely associated soluble NR activity.

III. Genetic Engineering of Kembrane Associated Nitrate Reductase This invention further comprises the gene sequences coding for membrae associated nitrate reductase, expression vehicles containing the gene sequence, hosts transformed therewith, the nitrate reductase produced by such transformed host expression, and uses for the nitrate reductase.

Any of a variety of procedures may be used to clone the nitrate reductase-encoding gene sequence. One such method entails analyzing a shuttle vector library of cDNA inserts (derived from a nitrate reductase expressing cell) for the presence of an insert which contains the nitrate reductase gene sequence. Such an analysis may be conducted by transfecting cells with the vector and then assaying for NR expression. One method for cloning the nitrate reductase gene sequence entails determining the amino acid sequence of the enzyme molecule. To accomplish this task, NR protein may be purified (using the assays described above), and analyzed to determine the amino acid sequence of the protein. Any method capable of elucidating such a sequence can be employed, I WO 91/04325 PCT/US90/04397 -12however, Edman degradation is preferred. The use of automated sequenators is especially preferred.

Although it is possible to determine the entire amino acid sequence of the enzyme, it is preferable to determine the sequence of peptide fragments of the molecule, and to use such sequence data to prepare oligonucleotide probes which can be used to isolate the entire NR gene sequence. NR peptide fragments can be obtained by incubating the intact molecule with cyanogen bromide, or with proteases such as papain, chymotrypsin or trypsin (Oike, Y. et al., J. Biol. Chem.

257:9751-9758 (1982); Liu, C. et al. Int. J. Pept. Protein Res. 21:209-215 (1983)). If the peptides are greater than amino acids long, the sequence information is generally sufficient to permit one to clone a gene sequence such as that encoding nitrate reductase.

Using the amino acid sequence information, the DNA sequences capable of encoding them are examined in order to clone the gene sequence encoding the nitrate reductase.

Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid (Watson, J.D., In: Molecular Biology of the Gene sequence, 3rd Ed., W.A.

Benjamin, Inc., Menlo Park, CA (1977), pp. 356-357).

Although occasionally an amino acid sequence may be encoded by only a single oligonucleotide, frequently the amino acid sequence may be encoded by any of a set of similar oligonucleotides. Importantly, whereas all of the members of this set contain oligonucleotides which are capable of encoding the peptide fragment and, thus, potentially contain the same oligonucleotide sequence as the gene sequence which encodes the peptide fragment, only one member of the set contains the nucleotide sequence that is identical to the nucleotide sequence of the gene sequence. Because this member is present within the set, and is capable of hybridizing to DNA even in the presence of the other members of the set, it WO 91/04325 PCT/US90/04397 -13is possible to employ the unfractionated set of oligonucleotides in the same manner in which one would employ a single oligonucleotide to clone the gene sequence that encodes the peptide.

Using the genetic code (Watson, In: Molecular Biology of the Gene sequence, 3rd Ed., W.A. Benjamin, Inc., Menlo Park, CA (1977)), one or more different oligonucleotides can be identified, each of which would be capable of encoding the NR peptides. The probability that a particular oligonucleotide will, in fact, constitute the actual nitrate reductase encoding sequence can be estimated by considering abnormal base pairing relationships and the frequency with which a particular codon is actually used (to encode a particular amino acid) in eukaryotic cells. Such "codon usage rules" are disclosed by Lathe, et al., J. Molec. Biol.

183:1-12 (1985). Using the "codon usage rules" of Lathe, a single oligonucleotide, or a set of oligonucleotides, that contains a theoretical "most probable" nucleotide sequence capable of encoding the NR peptide sequences is identified.

The oligonucleotide, or set of oligonucleotides, containing the theoretical "most probable" sequence capable of encoding the nitrate reductase gene sequence fragments is used to identify the sequence of a complementary oligonucleotide or set of oligonucleotides which is capable of hybridizing to the "most probable" sequence, or set of sequences. An oligonucleotide containing such a complementary sequence can be employed as a probe to identify and isolate the NR gene sequence (Maniatis, et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1982).

Thus, in summary, the actual identification of NR peptide sequences permits the identification of a theoretical "most probable" DNA sequence, or a set of such sequences, capable of encoding such a peptide. By constructing an oligonucleotide WO 91/04325 PCT/US90/04397 -14complementary to this theoretical sequence (or by constructing a set of oligonucleotides complementary to the set of "most probable" oligonucleotides), one obtains a DNA molecule (or set of DNA molecules), capable of functioning as a probe to identify and isolate the NR gene sequence.

The process for genetically engineering the nitrate reductase according to the invention, is facilitated through the cloning of genetic sequences which are capable of encoding the nitrate reductase and through the expression of such genetic sequences. As used herein, the term "gene sequence" is intended to refer to a nucleic acid molecule (preferably DNA). Gene sequences which are capable of encoding the nitrate reductase may be derived from a variety of sources.

These sources include genomic DNA, cDNA, synthetic DNA, and combinations thereof.

Genomic DNA may or may not include naturally occurring introns. Moreover, such genomic DNA may be obtained in association with the 5' promoter region of the NR gene sequences. To the extent that a host cell can recognize the transcriptional regulatory and translational initiation signals associated with the expression of the protein, then the region 5' may be retained and employed for transcriptional and translational initiation regulation.

For cDNA, the cDNA may be cloned and the resulting clone screened with an appropriate probe for cDNA coding for the desired sequences. Once the desired clone has been isolated, the cDNA may be manipulated in substantially the same manner as the genomic DNA. However, with cDNA there will be no "A introns or intervening sequences. For this reason, a cDNA molecule which encodes the nitrate reductase is the preferred gene sequence of the present invention.

Genomic DNA or cDNA may be obtained in several ways.

Genomic DNA can be extracted and purified from suitable cells by means well known in the art. Alternatively, mRNA can be I I WO 91/04325 PCT/US90/04397 isolated from a cell which produces the nitrate reductase and used to produce cDNA by means well known in the art. Such suitable DNA preparations are enzymatically cleaved, or randomly sheared, and ligated into recombinant vectors to form a gene sequence library. Such vectors can then be screened with the above-described oligonucleotide probes in order to identify a NR encoding sequence.

A suitable oligonucleotide, or set of oligonucleotides, which is capable of encoding a fragment of the NR gene sequence (or which is complementary to such an oligonucleotide, or set of oligonucleotides) identified using the above-described procedure, is synthesized, and hybridized by means well known in the art, against a DNA or, more preferably, a cDNA preparation, derived from cells which are capable of expressing the NR gene sequence. The source of DNA or cDNA used will preferably have been enriched for NR sequences. Such enrichment can most easily be obtained from cDNA obtained by extracting RNA from cells which produce high levels of the NR gene sequence. Techniques of nucleic acid hybridization are disclosed by Maniatis, et al. (In: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY (1982)), and by Haymes, et al. (In: Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC (1985)), which references are herein incorporated by reference.

To facilitate the detection of the desired NR encoding sequence, the above-described DNA probe may be labeled with a deteztable group. Such detectable group can be any material having a detectable physical or chemical property. Such materials have been well-developed in the field of immunoassays and in general most any label useful in such methods can be applied to the present invention. Particularly useful are enzymatically active groups, such as enzymes (see Clin. Chem. 22:1243 (1976)), enzyme substrates (see British IL WO 91/04325 PCT/US90/04397 -16- Pat. Spec. 1,548,741), coenzymes (see U.S. Pat. Nos. 4,230,797 and 4,238,565) and enzyme inhibitors (see U.S. Pat. No.

4,134,792;; fluorescers (see Clin. Chem. 25:353 (1979)); chromophores; luminescers such as chemiluminescers and bioluminescers (see Clin. Chem. 5:512 (1979)); specifically bindable ligands; proximal interacting pairs; and radioisotopes such as 3 H, 35 S, 32 p, 1251 and 14 C. Such labels and labeling pairs are detected on the basis of their own physical properties fluorescers, chromophores and radioisotopes) or their reactive or binding properties enzymes, substrates, coenzymes and inhibitors). For example, a cofactor-labeled probe can be detected by adding the enzyme for which the label is a cofactor and a substrate for the enzyme. For example, one can use an enzyme which acts upon a substrate to generate a product with a measurable physical property. Examples of the latter include, but are not limited to, beta-galactosidase, alkaline phosphatase and peroxidase.

Those members of the above-described gene sequence library which are found to be capable of such hybridization are then analyzed to determine the extent and nature of the NR encoding sequences which they contain.

In an alternative way of cloning the NR gene sequence, a library of expression vectors is prepared by cloning DNA or, more preferably cDNA, from a cell capable of expressing NR into an expression vector. The library is then screened for members capable of expressing a protein which binds to anti-NR antibody, and which has a nucleotide sequence that is capable of encoding polypeptides that have the same amino acid sequence as the NR or fragments of the NR.

The cloned nitrate reductase encoding sequences, obtained through the methods described above, may be operably linked to an expression vector, and introduced into bacterial, or eukaryotic cells to produce NR, or a functional derivative WO 91/04325 PCT/US90/04397 -17thereof. Techniques for such manipulations are disclosed by Maniatis, T. et al., supra, and are well known in the art.

The above discussed methods are, therefore, capable of identifying a gene sequence which is capable of encoding the nitrate reductase or fragments thereof. In order to further characterize such a gene sequence, it is desirable to express the NR which such a sequence encodes, and confirm that it possesses the characteristics of the NR. Such characteristics may include the ability to specifically bind anti-NR antibody, the ability to elicit the production of antibody which are capable of binding to the NR, the ability to provide nitrate reductase activity to a recipient cell, etc.

In lieu of using the above-described recombinant methods, a gene sequence which encodes the NR can be prepared by synthetic means (such as by organic synthetic means, etc.) IV. Expression of the Nitrate Reductase and its Functional Derivatives.

The nitrate reductase encoding sequences, obtained tirough the methods described above, may be operably linked to an expression vector, and introduced into prokaryotic or eukaryotic cells in order to produce the nitrate reductase or its functional derivatives. The present invention pertains both to the intact nitrate reductase and to the functional derivatives of this nitrate reductase. A "functional derivative" of the nitrate reductase is a compound which possesses a biological activity (either functional or structural) that is substantially similar to a biological activity of the nitrate reductase. The term "functional derivative" is intended to include the "fragments," "variants," "analogues," or "chemical derivatives" of a molecule. A "fragment" of a molecule such as the nitrate

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WO 91/04325 PCI/US90/04397 -18reductase is meant to refer to any polypeptide subset of the molecule. A "variant" of a molecule such as the nitrate reductase is meant to refer to a molecule substantially similar in structure and function to either the entire molecule, or to a fragment thereof. A molecule is said to be "substantially similar" to another molecule if both molecules have substantially similar structures or if both molecules possess a similar biological activity. Thus, provided that two molecules possess a similar activity, they are considered variants as that term is used herein even if the structure of one of the molecules is not found in the other, or if the sequence of amino acid residues is not identical. An "analog" of a molecule such as the nitrate reductase is meant to refer to a molecule substantially similar in function to either the entire molecule or to a fragment thereof. As used herein, a molecule is said to be a "chemical derivative" of another molecule when it contains additional chemical moieties not normally a part of the molecule. Such moieties may improve the molecule's solubility, absorption, biological half life, etc. Moieties capable of mediating such effects are disclosed in Remington's Pharmaceutical Sciences (1980). Procedures for coupling such moieties to a molecule are well known in the art.

A DNA sequence encoding the nitrate reductase or its functional derivatives may be recombined with vector DNA in accordance with conventional techniques, including blunt-ended or staggered-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Techniques for such manipulations are disclosed by Maniatis, et al., supra, and are well known in the art.

A nucleic acid molecule, such as DNA, is said to be "capable of expressing" a polypeptide if it contains WO 91/04325 PCT/US90/04397 -19nucleotide sequences which contain transcriptional and translational regulatory information and such sequences are "operably linked" to nucleotide sequences which encode the polypeptide. An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed are connected in such a way as to permit gene sequence expression. The precise nature of the regulatory regions needed for gene sequence expression may vary from organism to organism, but shall in general include a promoter region which, in prokaryotes, contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal the initiation of the nitrate reductase synthesis.

Such regions will normally include those sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and the like.

If desired, the non-coding region 3' to the gene sequence coding for the nitrate reductase may be obtained by the abovedescribed methods. This region may be retained for its transcriptional termination regulatory sequences, such as termination and polyadenylation. Thus, by retaining the 3'region naturally contiguous to the DNA sequence coding for the nitrate reductase, the transcriptional termination signals may be provided. Where the transcriptional termination signals are not satisfactorily functional in the expression host cell, then a 3' region functional in the host cell may be substituted.

Two DNA sequences (such as a promoter region sequence and the nitrate reductase encoding sequence) are said to be operably linked if the nature of the linkage between the two DNA sequences does not result in the introduction of a frame-shift mutation, interfere with the ability of the promoter region sequence to direct the transcription of the WO 91/04325 PCT/US90/04397 nitrate reductase gene sequence, or interfere with the ability of the nitrate reductase gene sequence to be transcribed by the promoter region sequence. Thus, a promoter region would be operably linked to a DNA sequence if the promoter were capable of effecting transcription of that DNA sequence.

Thus, to express the nitrate reductase transcriptional and translational signals recognized by an appropriate host are necessary.

The present invention encompasses the expression of the nitrate reductase protein (or a functional derivative thereof) in either prokaryotic or eukaryotic cells. Preferred prokaryotic hosts include bacteria such as E. coli, Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, etc. The most preferred prokaryotic host is E. coli. Bacterial hosts of particular interest include E. coli K12 strain 294 (ATCC 31446), E. coli X1776 (ATCC 31537), E. coli W3110 lambdaprototrophic (ATCC 27325)), and other enterobacterium such as Salmonella typhimurium or Serratia marcescens, and various Pseudomonas species. Under such conditions, the nitrate reductase will not be glycosylated. The procaryotic host must be compatible with the replicon and control sequences in the expression plasmid.

To express the nitrate reductase (or a functional derivative thereof) in a prokaryotic cell (such as, for example, E. coli, B. subtilis, Pseudomonas, Streptomyces, etc.), it is necessary to operably link the nitrate reductase encoding sequence to a functional prokaryotic promoter. Such promoters may be either constitutive or, more preferably, regulatable inducible or derepressible). Examples of constitutive promoters include the int promoter of bacteriophage X, the bla promoter of the P-lactamase gene sequence of pBR322, and the CAT promoter of the chloramphenicol acetyl transferase gene sequence of pPR325, WO 91/04325 PCT/US90/04397 -21etc. Examples of inducible prokaryotic promoters include the major right and left promoters of bacteriophage X (PL and PR), the trp, recA, lacZ, lacI, and gal promoters of E. coli, the a-amylase (Ulmanen, et al., J. Bacteriol. 162:176-182 (1985)) and the a-28-specific promoters of B. subtilis (Gilman, et al., Gene sequence 32:11-20 (1984)), the promoters of the bacteriophages of Bacillus (Gryczan, T.J., In: The Molecular Biology of the Bacilli, Academic Press, Inc., NY (1982)), and Streptomyces promoters (Ward, et al., Mol. Gen. Genet. 203:468-478 (1986)). Prokaryotic promoters are reviewed by Glick, Ind. Microbiol.

1:277-282 (1987)); Cenatiempo, Y. (Biochimie 68:505-516 (1986)); and Gottesman, S. (Ann. Rev. Genet. 18:415-442 (1984)).

Proper expression in a prokaryotic cell also requires the presence of a ribosome binding site upstream of the gene sequence-encoding sequence. Such ribosome binding sites are disclosed, for example, by Gold, et al. (Ann. Rev.

Microbiol. 35:365-404 (1981)).

Preferred eukaryotic hosts include yeast, fungi, insect cells, mammalian cells either in vivo, or in tissue culture.

Mammalian cells which may be useful as hosts include cells of fibroblast origin such as VERO or CHO-K1, or cells of lymphoid origin, such as the hybridoma SP2/0-AG14 or the myeloma P3x63Sg8, and their derivatives. Preferred mammalian host cells include SP2/0 and J558L, as well as neuroblastoma cell lines such as IMR 332 that may provide better capacities for correct post-translational processing.

For a mammalian host, several possible vector systems are available for the expression of the nitrate reductase. A wide variety of transcriptional and translational regulatory sequences may be employed, depending upon the nature of the host. The transcriptional and translational regulatory signals may be derived from viral sources, such as adenovirus, I

-A-

WO 91/04325 PCT/US90/04397 -22bovine papilloma virus, Simian virus, or the like, where the regulatory signals are associated with a particular gene sequence which has a high level of expression.

Alternatively, promoters from mammalian expression products, such as actin, collagen, myosin, etc., may be employed.

Transcriptional initiation regulatory signals may be selected which allow for repression or activation, so that expression of the gene sequences can be modulated. Of interest are regulatory signals which are temperature-sensitive so that by varying the temperature, expression can be repressed or initiated, or are subject to chemical (such as metabolite) regulation.

Yeast provides substantial advantages in that it can also carry out post-translational peptide modifications. A number of recombinant DNA strategies exist which utilize strong promoter sequences and high copy number of plasmids which can be utilized for production of the desired proteins in yeast.

Yeast recognizes leader sequences on cloned mammalian gene sequence products and secretes peptides bearing leader sequences (i pre-peptides).

Any of a series of yeast gene sequence expression systems incorporating promoter and termination elements from the actively expressed gene sequences coding for glycolytic enzymes produced in large quantities when yeast are grown in mediums rich in glucose can be utilized. Known glycolytic gene sequences can also provide very efficient transcriptional control signals. For example, the promoter and terminator signals of the phosphoglycerate kinase gene sequence can be utilized.

Another preferred host is insect cells, for example the Drosophila larvae. Using insect cells as hosts, the Drosophila alcohol dehydrogenase promoter can be used. Rubin, Science 240:1453-1459 (1988). Alternatively, baculovirus vectors can be engineered to express large amounts WO 91/04325 PCT/US90/04397 -23of the nitrate reductase in insects cells (Jasny, B.R., Science 238:1653 (1987); Miller, et al., in Genetic Engineering (1986), Setlow, et al., eds., Plenum, Vol.

8, pp. 277-297).

As discussed above, expression of the nitrate reductase in eukaryotic hosts requires the use of eukaryotic regulatory regions. Such regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis.

Preferred eukaryotic promoters include the promoter of the mouse metallothionein I gene sequence (Hamer, et al., J.

Mol. Appl. Gen. 1:273-288 (1982)); the TK promoter of Herpes virus (McKnight, Cell 31:355-365 (1982)); the SV40 early promoter (Benoist, et al., Nature (London) 290:304-310 (1981)); the yeast gal4 gene sequence promoter (Johnston, et al., Proc. Natl. Acad. S:i. (USA) 79:6971-6975 (1982); Silver, et al., Proc. Natl. Acad. Sci. (USA) 81:5951-5955 (1984)).

As is widely known, translation of eukaryotic mRNA is initiated at the codon which encodes the first methionine.

For this reason, it is preferable to ensure that the linkage between a eukaryotic promoter and a DNA sequence which encodes the nitrate reductase (or a functional derivative thereof) does not contain any intervening codons which are capable of encoding a methionine AUG). The presence of such codons results either in a formation of a fusion protein (if the AUG codon is in the same reading frame as the nitrate reductase encoding DNA sequence) or a frame-shift mutation (if the AUG codon is not in the same reading frame as the nitrate reductase encoding sequence).

The nitrate reductase encoding sequence and an operably linked promoter may be introduced into a recipient prokaryotic or eukaryotic cell either as a non-replicating DNA (or RNA) molecule, which may either be a linear molecule or, more preferably, a closed covalent circular molecule. Since such I WO 91/04325 PCT/US90/04397 -24molecules are incapable of autonomous replication, the expression of the nitrate reductase may occur through the transient expression of the introduced sequence. Alternatively, permanent expression may occur through the integration of the introduced sequence into the host chromosome.

In one embodiment, a vector is employed which is capable of integrating the desired gene sequences into the host cell chromosome. Cells which have stably integrated the introduced DNA into their chromosomes can be selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector. The marker may provide for prototrophy to an auxotrophic host, biocide resistance, antibiotics, or heavy metals, such as copper, or the like. The selectable marker gene sequence can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by co-transfection. Additional elements may also be needed for optimal synthesis of single chain binding protein mRNA. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals. cDNA expression vectors incorporating such elements include those described by Okayama, Molec.

Cell. Biol. 3:280 (1983).

In a preferred embodiment, the introduced sequence will be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host. Any of a wide variety of vectors may be employed for this purpose. Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species. Preferred WO 91/04325 PCT/US90/04397 prokaryotic vectors include plasmids such as those capable of replication in E. coli (such as, for example, pBR322, ColE1, pSC101, pACYC 184, wVX. Such plasmids are, for example, disclosed by Maniatis, et al. (In: Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1982)). Bacillus plasmids include pC194, pC221, pT127, etc. Such plasmids are disclosed by Gryczan, T. (In: The Molecular Biology of the Bacilli, Academic Press, NY (1982), pp. 307-329). Suitable Streptomyces plasmids include pIJ101 (Kendall, et al., J. Bacteriol. 169:4177-4183 (1987)), and streptomyces bacteriophages such as OC31 (Chater, et al., In: Sixth International Symposium on Actinomycetales Biology, Akademiai Kaido, Budapest, Hungary (1986), pp. 45-54). Pseudomonas plasmids are reviewed by John, et al. (Rev. Infect. Dis. 8:693-704 (1986)), and Izaki, K. (Jpn. J. Bacteriao. 33:729-742 (1978)).

Preferred eukaryotic plasmids include BPV, vaccinia, 2-micron circle, etc., or their derivatives. Such plasmids are well known in the art (Botstein, et al., Miami Wntr. Symp. 19:265-274 (1982); Broach, In: The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p. 445-470 (1981); Broach, Cell 28:203-204 (1982); Bollon, et al., J. Clin. Hematol. Oncol. 10:39- 48 (1980); Maniatis, In: Cell Bioloqy: A Comprehensive Treatise, Vol. 3, Gene sequence Expression, Academic Press, NY, pp. 563-608 (1980)).

Once the vector or DNA sequence containing the construct(s) has been prepared for expression, the DNA construct(s) may be introduced into an appropriate host cell by any oi a variety of suitable means: transformation, transfection, conjugation, protoplast fusion, electroporation, calcium phosphate-precipitation, direct microinjection, etc. After the introduction of the vector, WO 91/04325 P(7/US90/04397 -26recipient cells are grown in a selective medium, which selects for the growth of vector-containing cells. Expression of the cloned gene sequence(s) results in the production of the nitrate reductase, or in the production of a fragment of this nitrate reductase. This can take place in the transformed cells as such, or following the induction of these cells to differentiate (for example, by administration of bromodeoxyuracil to neuroblastoma cells or the like).

The expressed protein may be isolated and purified in accordance with conventional conditions, such as extraction, precipitation, chromatography, affinity chromatography, electrophoresis, or the like.

V. Uses of the Nitrate Reductase to Genetically Modify Plants The nitrate reductase gene sequences can be introduced into a plant by genetic engineering techniques, which upon production in the plant cell could be used as a means for facilitating the fixation of nitrogen by the plant, and therby lessen, or obviate, the plant's requirement for reduced nitrogen. Therefore, it is possible to produce a plant that is less dependent upon fertilizers, and more able to survive adverse culturing conditions. In thus another embodiment of this invention, the NR gene sequence is used to transform a plant to enhance or confer a nitrate reductase activity to the plant.

The coding region for an NR gene sequence that may be used in this invention may be the full-length or partial active length of the gene sequence. It is necessary, however, that the genetic sequence coding for the nitrate reductase be expressed, and produced, as a functional nitrate reductase in the resulting plant cell.

1 WO 91/04325 PCT/US90/04397 -27- DNA from both genomic DNA and cDNA and synthetic DNA encoding a nitrate reductase gene sequence may be used in this invention. Further, a nitrate reductase gene sequence may be constructed partially of a cDNA clone, partially of a genomic clone, and partially of a synthetic gene sequence and various combinations thereof. In addition, the DNA coding for the nitrate reductase gene sequence may comprise portions from various species.

In another embodiment, this invention comprises chimeric gene sequences: a first gene sequence coding for a nitrate reductase that upon expression of the gene sequence in a given plant cell is functional for the nitrate reductase; one or more additional gene sequences operably linked on either side of the nitrate reductase coding region.

These additional gene sequences contain sequences for promoter(s) or terminator(s). The plant regulatory sequences may be heterologous or homologous to the host cell.

In a preferred embodiment, the promoter of the nitrate reductase gene sequence is used to express the chimeric gene sequence. Other promoters that may be used in the gene sequence include nos, ocs, and CaMV promoters. An efficient plant promoter that may be used is an overproducing plant promoter. This promoter in operable linkage with the gene sequence for the nitrate reductase should be capable of promoting expression of then nitrate reductase. Overproducing plant promoters that may be used in this invention include the promoter of the small subunit (ss) of the biphosphate carboxylase from soybean (Berry-Lowe et al., J.

Molecular and App. Gen., 1:483-498 (1982), and the promoter of the chlorophyll a/b binding protein. These two promoters are known to be light induced in eukaryotic plant cells (see, for example, Genetic Engineering of Plants, an Agricultural Perspective, A. Cashmore, Plenum, New York 1983, pages 29-38; WO 91/04325 PCr/US90/04397 -28- Corruzi, G. et al., J. of Biol. Chem., 258: 1399 (1983); and Dunsmuir, P. et al., J. of Mol. and Applied Genet., 2: 285 (1983)).

Further, in another preferred embodiment, the expression of the chimeric gene sequence comprising the nitrate reductase gene sequence is operably linked in correct reading frame with a plant promoter and with a gene sequence secretion signal sequence.

The chimeric gene sequence comprising a nitrate reductase gene sequence operably linked to a plant promoter, and in the preferred embodiment with the secretion signal sequences, can be ligated into a suitable cloning vector. In general, plasmid or viral (bacteriophage) vectors containing replication and control sequences derived from species compatible with the host cell are used. The cloning vector will typically carry a replication origin, as well as specific gene sequences that are capable of providing phenotypic selection markers in transformed host cells, typically resistance to antibiotics. The transforming vectors can be selected by these phenotypic markers after transformation in a host cell.

Host cells that may be u ad in this invention include procaryotes, including bacterial hosts such as E. coli, S typhimurium, and Serratia marcescens. Eukaryotic hosts such as yeast or filamentous fungi may also be used in this invention.

The cloning vector and host cell transformed with the vector are used in this invention typically to increase the copy number of the vector. With an increased copy number, the vectors containing the nitrate reductase gene sequence can be isolated and, for example, used to introduce the chimeric gene sequences into the plant cells. The genetic material contained in the vector can be microinjected directly into plant cells by use of micropipettes to mechanically transfer WO 91/04325 PCT/US90/04397 -29the recombinant DNA. The genetic material may also be transferred into the plant cell by using polyethylene glycol which forms a precipitation complex with the genetic material that is taken up by the cell. (Paszkowski et al., EMBO J.

3:2717-22 (1984)).

In an alternative embodiment of this invention, the nitrate reductase gene sequence may be introduced into the plant cells by electroporation. (Fromm et al., "Expression of Gene sequences Transferred into Monocot and Dicot Plant Cells by Electroporation," Proc. Natl. Acad. Sci. U.S.A. 82:5824 (1985)). In this technique, plant protoplasts are electroporated in the presence of plasmids containing the nitrate reductase genetic construct. Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the plasmids. Electroporated plant protoplasts reform the cell wall, divide, and form plant callus. Selection of the transformed plant cells with the expressed nitrate reductase can be accomplished using the phenotypic markers as described above.

Another method of introducing the nitrate reductase gene sequence into plant cells is to infect a plant cell with Agrobacterium tumefaciens transformed with the nitrate reductase gene sequence. Under appropriate conditions known in the art, the transformed plant cells are grown to form shoots, roots, and develop further into plants. The nitrate reductase gene sequence can be introduced into appropriate plant cells, for example, by means of the Ti plasmid of Aqrobacterium tumefaciens. The Ti plasmid is transmitted to plant cells on infection by Aqrobacterium tumefaciens and is stably integrated into the plant genome. Horsch et al., "Inheritance of Functional Foreign Genes in Plants," Science 233:496-498 (1984); Fraley et al., Proc. Natl. Acad. Sci.

U.S.A. 80:4803 (1983)).

WO 91/04325 PCT/US90/04397 Ti plasmids contain two regions essential for the production of transformed cells. One of these, named transfer DNA (T DNA), induces tumor formation. The other, termed virulent region, is essential for the formation but not maintenance of tumors. The transfer DNA region, which transfers to the plant genome, can be increased in size by the insertion of the enzyme's gene sequence without its transferring ability being affected. By removing the tumorcausing gene sequences so that they no longer interfere, the modified Ti plasmid can then be used as a vector for the transfer of the gene sequence constructs of the invention into an appropriate plant cell.

All plant cells which can be transformed by Aqrobacterium and whole plants regenerated from the transformed cells can also be transformed according to the invention so to produce transformed whole plants which contain the transferred nitrate reductase gene sequence.

There are presently two different ways to transform plant cells with Aqrobacterium: co-cultivation of Aqrobacterium with cultured isolated protoplasts, or transforming cells or tissues with Aqrobacterium.

Method requires an established culture system that allows culturing protoplasts and plant regeneration from cultured protoplasts.

Method requires that the plant cells or tissues can be transformed by Aqrobacterium and that the transformed cells or tissues can be induced to regenerate into whole plants. In the binary system, to have infection, two plasmids are needed: a T-DNA containing plasmid and a vir plasmid.

After transformation of the plant cell or plant, those plant cells or plants transformed by the Ti plasmid so that the enzyme is expressed, can be selected by an appropriate WO 91/04325 PCT/US90/04397 -31phenotypic marker. These phenotypical markers include, but are not limited to, antibiotic resistance. Other phenotypic markers are known in the art and may be used in this invention.

All plants from which protoplasts can be isolated and cultured to give whole regenerated plants can be transformed by the present invention so that whole plants are recovered which contain the transferred nitrate reductase gene sequence.

Some suitable plants include, for example, species from the genera Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manicot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersion, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Hemerocallis, Nemesia, Pelargonium, Panicum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browallia, Glycine, Lolium, Zea, Triticum, Sorghum, and Datura.

There is an increasing body of evidence that practically all plants can be regenerated from cultured cells or tissues, including but not limited to all major cereal crop species, sugarcane, sugar beet, cotton, fruit and other trees, legumes and vegetables. Limited knowledge presently exists on whether all of these plants can be transformed by Aqrobacterium.

Species which are a natural plant host for Agrobacterium may be transformable in vitro. Monocotyledonous plants, and in particular, cereals and grasses, are not natural hosts to Agrobacterium. Attempts to transform them using Aqrobacterium have been unsuccessful until recently. Hooykas-Van Slogteren et al., Nature 311:763-764 (1984). There is growing evidence now that certain monocots can be transformed by Agrobacterium.

Using novel experimental approaches that have now become available, cereal and grass species may be transformable.

F~I~ ~__111~ WO 91/04325 PCT/ US90/04397 -32- Additional plant genera that may be transformed by Aqrobacterium include Ipomoea, Passiflora, Cyclamen, Malus, Prunus, Rosa, Rubus, Populus, Santalum, Allium, Lilium, Narcissus, Ananas, Arachis, Phaseolus, and Pisum.

Plant regeneration from cultural protoplasts is described in Evans et al., "Protoplast Isolation and Culture," in Handbook of Plant Cell Culture 1:124-176 (MacMillan Publishing Co., New York, 1983); M.R. Davey, "Recent Developments in the Culture and Regeneration of Plant Protoplasts," Protoplasts, 1983 Lecture Proceedings, pp. 19-29 (Birkhauser, Basel, 1983); P.J. Dale, "Protoplast Culture and Plant Regeneration of Cereals and Other Recalcitrant Crops," in Protoplasts 1983 Lecture Proceedings, pp. 31-41 (Birkhauser, Basel. 1983); and H. Binding, "Regeneration of Plants," in Plant Protoplasts, pp. 21-37 (CRC Press, Boca Raton, 1985).

Regeneration varies from species to species of plants, but generally a suspension of transformed protoplasts containing multiple copies of the nitrate reductase gene sequence is first provided. Embryo formation can then be induced from the protoplast suspensions, to the stage of ripening and germination as natural embryos. The culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. It is also advantageous to add glutamic acid and proline to the medium, especially for such species as corn and alfalfa. Shoots and roots normally develop simultaneously. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is fully reproducible and repeatable.

The mature plants, grown from the transformed plant cells, are selfed to produce an inbred plant. The inbred plant produces seed containing the gene sequence for the nitrate reductase. These seeds can be grown to produce plants that have the nitrate reductase.

WO 91/04325 PCT/US90/04397 -33- The inbreds according to this invention can be used to develop insect tolerant hybrids. In this method, an insect tolerant inbred line is crossed with another inbred line to produce the hybrid.

Parts obtained from the regenerated plant, such as flowers, seeds, leaves, branches, fruit, and the like are covered by the invention provided that these parts comprise the insect tolerant cells. Progeny and variants, and mutants of the regenerated plants are also included within the scope of this invention.

In diploid plants, typically one parent may be transformed by the nitrate reductase gene sequence and the other parent is the wild type. After crossing the parents, the first generation hybrids (Fl) will show a distribution of 1/2 nitrate reductase/wild type:1/2 nitrate reductase/wild type. These first generation hybrids (Fl) are selfed to produce second generation hybrids The genetic distribution of the F2 hybrids are 1/4 nitrate reductase/nitrate reductase 1/2 nitrate reductase/wild type 1/4 wild type/wild type. The F2 hybrids with the genetic makeup of nitrate reductase/nitrate reductase are chosen as the herbicidal tolerant plants.

As used herein, variant describes phenotypic changes that are stable and heritable, including heritable variation that is sexually transmitted to progeny of plants, provided that the variant still comprises an insect tolerant plant. Also, as used herein, mutant describes variation as a result of environmental conditions, such as radiation, or as a result of genetic variation in which a trait is transmitted meiotically according to well-established laws of inheritance. The mutant plant, however, must still exhibit an insect tolerance according to the invention.

Having now fully described the invention, the same will be more clearly understood by reference to specific examples

A

WO 91/04325 PCT/US90/04397 -34which are intended to be illustrative, and not limiting of the invention, unless indicated.

EXAMPLE 1 PREPARATION OF CORN-DERIVED MATERIALS Plant material. Corn seedlings (Zea mays Golden Jubilee) were grown hydroponically. Seeds (kernels) were soaked in volume/volume NaOC1 prepared from common bleach for min, rinsed with distilled water and germinated at room temperature in aerated distilled water. After 24 h, the germinated seeds were spread on a stainless steel screen about I cm above the surface of 5 1 of aerated 0.2 mM CaC12 solution, covered with plastic wrapping and placed in the dark at 25'C. The plastic wrapping was removed after 3 d.

After 5 d the seedlings were transferred to aerated 0.1strength Hoagland solution no. 1, lacking N (Hoagland, D.R., and Arnon, Cal. Agric. Exp. Stat. Circ. No. 347 (1950)) and placed in a controlled environment growth chamber. The growth conditions were 60-65% relative humidity, 25'C, and continuous light to minimize the effect of rhythms on NRA levels. The photon flux density at the seedling canopy was 400 pmol.m- 2 .s 1 measured at 400-700 nm with a Licor 190 S sensor and was supplied by incandescent and cool white fluorescent lamps, respectively, at a ratio of 1:5. Lights were obtained from Sylvania Lighting System, Danvers, MA.

After 2 d in continuous light the seedlings were transferred to 0.1 strength Hoagland solution no. 1 containing 1 mM KNO 3 for 24 h to induce NRA.

Microsome isolation. Roots (25 g) from 9-d-old corn seedlings were excised just below the seed, snipped into 1 cm pieces with a pair of scissors, and then ground in 60 ml of cold grinding buffer in a chilled mortar and pestle. The grinding WO 91/04325 PCT/US90/04397 buffer consisted of 250 mM sucrose, 3 mM ethylenediaminetetraacetic acid (EDTA), 50 mM 2-amino-2- (hydroxymethyl)-1,3-propanediol (Tris-HCl) (pH 0.05% (w/w roots) polyvinylpolypyrrolidone, 1 mM dithiothreitol (DTT), 3 mM phenylmethylsulfonyl fluoride (PMSF), 1 AM Na 2 MoO 4

(H

2 0) 2 and 5 pM flavine-adenine dinucleotide (FAD).

The PMSF was made up as a 0.1 M stock in isopropanol and added at a ratio of 0.75 ml PMSF: 25 ml grind buffer. The DTT was made up as a 1.0 M stock in deionized water and added at a ratio of 0.025 ml DTT: 25 ml grind buffer. All chemicals used in the grind buffer were obtained from Sigma Chemical Co., St. Louis, MO, USA. The homogenate was filtered through 4 layers of cheesecloth and centrifuged at 12,000.g for min. In some experiments a portion of the post-12,000.g fraction was saved for enzyme analysis. The supernatant was centrifuged at 120,000.g for 20 min. The resulting pellet represented a microsomal fraction, a crude membrane fraction. The post-120,000.g fraction was used in some experiments for enzyme analysis.

For determinations of NRA, microsomal pellets were suspended in resuspension buffer, diluted to 20 ml, and centrifuged at 120,000.g for 20 min. The resuspension buffer was 2 mM Tris-HC1 (pH 1 iM Na 2 MoO 4

(H

2 0) 2 5 IM FAD, 3 mM PMSF and 1 mM DTT. The process was repeated three more times, and samples from each membrane pellet including the initial microsome fraction were saved to determine NRA.

Plasma-membrane isolation. Microsomal pellets were resuspended in 2.2 ml of 250 mM sucrose, 5 mM K-phosphate (pH 7.8) and 1 mM PMSF, and partitioned in an aqueous polymer twophase system as detailed by Larsson et al. (Plasma Membranes, In: Modern Methods of Plant Analysis, New Series, Vol. 1, Cell Components, HF Linksens, et al., eds., Springer-Verlag, Berlin, pp. 85-104 (1985)). All steps in the procedure were WO 91/04325 PCT/US90/04397 -36carried out at 4'C. Briefly, 2 ml of the resuspended microsome fraction was added to an 8 ml two-phase system that contained 6.5% Dextran T 500 (Pharmacia Fine Chemicals, Piscataway, NJ, USA), 6.5% polyethylene gl1,col 3350 (Sigma Chemical Co., St. Louis, MO, USA), 0.33 M sucrose, 3 mM KC1 and 5 mM K-phosphate (pH The contents of the tubes was mixed by inversion 40-50 times, and centrifuged in a swinging-bucket rotor at 1,200.g for 5 min. The upper phase was collected and repartitioned on a fresh lower phase as described by Sandstrom et al. (Sandstrom, et al., Plant Physiol. 85:693-698 (1987)). The remaining microsome, U 2 and the first lower phase (L 1 fractions were collected and each was diluted with 20 ml of resuspension buffer and centrifuged at 120,000.g for 20 mii. The U 2 pellet was suspended in resuspension buffer, diluted to 20 ml and again centrifuged at 120,000.g for 20 min. The resulting pellets were suspended and diluted to a protein concentration equivalent to the post- 120,000.9 fraction in resuspension buffer, and used directly for enzyme analysis.

Salt washes. Twice-centrifuged U 2 fractions were diluted to mg.ml._ protein concentration in resuspension buffer and brought to a final concentration of 0-1.0 M NaC1 in 0.5 ml.

All fractions were vigorously mixed for 30 sec and placed on ice. The salt treated fractions were sonicated on ice in an American Brand (American Scientific Product, McGraw Park, IL, USA) sonicator. After 20 min, the fractions were diluted with 8 ml of resuspension buffer and centrifuged at 150,000.g for min. Nitrate reductase activities were determined in the resuspended pellets.

Solubilization with Triton X-100. Twice-pelleted U 2 fractions were diluted to 0.5 mg.ml.I protein with resuspension buffer, brought to 0 to 0.1% Triton X-100 (octyl phenoxy WO 91/04325 PCT/US90/04397 -37polyethoxyethanol, Sigma Chemical Co., St. Louis, MO, USA) in a total volume of 0.5 ml, mixed vigorously for 30 s and then placed on ice. After 15 min, fractions were centrifuged at 150,000.g for 15 min without dilution. Vanadate-sensitive ATPase activity and NRA were determined in the resuspended pellets and the soluble fractions.

EXAMPLE 2 MEMBRANE-ASSOCIATED NR ACTIVITY IN CORN Nitrate reductase activity was detected in a crude membrane fraction isolated from corn roots. To release potentially trapped soluble NRA, the membrane pellet was washed four times in hypotonic buffer (Pupillo, and Del Grosso, Planta 151:506-511 (1981)). The initial wash released 23% of the NRA to the soluble fraction. This probably represented soluble NRA that was loosely bound or trapped within membrane vesicles. Subsequent washes of the membrane pellet released little additional NRA. This indicated that NRA detected in the washed membrane pellets was membrane-associated. In all subsequent experiments the membrane fractions were washed at least two times with hypotonic buffer to remove loosely associated soluble NRA.

The effect of Triton X-100 on both soluble and membraneassociated NR activity is shown in Table 1. Microsomal NR activity was stimulated up to nine fold by Triton X-100 while soluble NR activity was hardly affected. Maximum activity was reached at a Triton X-100 concentration of 0.043% which represented a T:p ratio of approx. 3.5 mg.mg-1. Higher Triton X-100 concentrations had little additional effect on the microsomal NR activity. Since the detergent activation was much greater for the membrane-associated NR activity than for the soluble NR activity, the stimulation by Triton X-100 was not the consequence of a general effect on NR activity but r WO 91/04325 PCT/US90/04397 -38appeared to be a specific activation of the membraneassociated enzyme. Similarly, particulate cellulase is stimulated by Triton X-100 while soluble cellulase is little affected (Koehler, et al., Plant Physiol. 58:324-330 (1976)).

For Table 1, corn seedlings were grown and root fractions were isolated as described above. The microsome fraction was pelleted and resuspended three times in hypotonic buffer, diluted to a protein concentration equivalent to the post 120,000 g(soluble) fraction (0.7 mg.ml- 1 and used directly for NR activity determination. Nitrate reductase activity was determined in the presence of increasing concentrations of Triton X-100 in the soluble fraction and the final microsomal pellet. The experiment was repeated three times and means SE are shown.

TABLE 1 Effect of Triton X-100 on soluble and microsomal NR activity from corn roots Nitrate Reduct se Activity (nmol.mg' .h 1 Triton X-100 T:p in (mg.mg- 1 Soluble Microsome Reaction Mixture 0 0 181 13 13.7 4 0.013 1.0 188 15 28.4 3 0.017 1.4 212 12 101 8 0.043 3.5 193 13 124 0.086 6.9 212 16 133 9 0.129 10.4 205 13 128 8 WO 91/04325 PCT/US90/04397 -39- EXAMPLE 3 LOCALIZATION OF MEMBRANE-ASSOCIATED CORN NR ACTIVITY Preliminary analysis of corn root microsome fractions by different. 11 sucrose gradient centrifugation indicated that microsome-associated NR activity was enriched in PM fractions.

In order to show that NR activity was localized in the PM, it was important to isolate highly purified PM fractions. It was especially important that the PM fractions be free of cytoplasmic material since, as explained in the Introduction, NR activity is generally considered to be localized in the cytosol.

Aqueous two-phase partitioning was used to isolate a PM fraction of very high purity from corn roots (Larsson, C.H., in Modern Methods of Plant Analysis, N.S. Series, Cell Components, vol. 1, pp. 85-104, Linskens, et al., eds., Springer-Verlag, Berlin, Heidelberg, New York (1985); Hodges, and Mills, Academic Press 118:41-54 (1986)).

Marker enzyme assessment of aqueous two-phase partitioned corn-root microsome fractions is shown in Table 2. A 2.3 fold enrichment of the PM H+-ATPase activity was detected in the U 2 fraction. Evaluation of markers for the tonoplast, vacuole, Golgi apparatus, endoplasmic reticulum, plastid and cytoplasm demonstrated that each of these activities was significantly reduced in U 2 Nitrate-sensitive ATPase activity (tonoplast) was reduced by 100%, NADH Cyt c reductase activity (endoplasmic reticulum) by 95%, latent UDPase activity (Golgi apparatus) by 96% and cytochrome c oxidase activity (mitochondria) by 91% in the U 2 fraction compared to the microsome. The lack of detectable plastid and cytosolic (Anderson, L.E. et al., Plant Phvsiol. 45:583-585 (1970); Miflin, B.J. et al., Plant Phvsiol. 53:870-74 (1974)) triose phosphate-isomerase activity and cytosolic (Quail, Annu.

Rev. Plant Phvsiol. 30:425-85 (1979)) alcohol dehydrogenase WO 91/04325 PCT/US90/04397 activity in U 2 provides evidence that this fraction was devoid of cytoplasmic contamination. The marker-enzyme data indicate that U 2 consists mainly of PM.

For Table 2, corn root fractions were isolated as described above. Preparations of the above fractions were used immediately after isolation to preserve activity and it was not possible to isolate a sufficient amount of the PM fraction to complete all the assays at one time; hence, several different preparations were required to complete all of the assays. Each of the assays was repeated at least three times using three different preparations providing the various fractions. The means SE are shown.

WO 91/04325 WO 9104325PCT/US90/04397 -41- TABLE 2 Marker Enzyme Assessment of Corn Root Fractions Activity Enzyme Phase /Plant Soluble Microsome Ll U 2 Fraction V an adate sensitive AT Pas5e a /PM 3 0.4 44.7 1.0 36.6 1.0 101 1.2

MO

3 sensitive AT Pasea /tonoplast 0.7 0.05 0.06 0.01 0.23 0.02 0 0 Latent UDPasea /Golgi 0.6 0.05 29.4 0.5 63.4 0.8 1.05 0.2 NADH Cyt reductase 0 /ER 0.08 0.01 0.9 0.05 1 0.1 0.05 0.005 Cyt c oxidase /miitochondria 0 0 0.3 0.05 0.5 0.05 0.03 0.005 TP 1 c /plastid /cytoplasm 5 0.5 5.5 0.6 9.1 0.5 0 0 ADHd /cytoplasm 52 1 0 0 0 0 0 0 a IAmol Pi.mg protein. h- 1 b pmol Cyt c.mg protein. h-I c Amol NADH.nig protein. h- 1 d nmol NAD.mg protein. h 1

I

WO 91/04325 PCT/US90/04397 -42- To determine if membrane associated NR activity was localized in the PM, the distribution of NR activity is soluble and phase-partitioned membrane fractions was determined (Table Approximately 1.8 19% of the total root NR activity (based upon the post 12,000 g supernatant NR activity) was detected in the microsome fraction in the absence and presence of Triton X-100, respectively. A 1.7- 2.6 fold enrichment of the NR activity in the U 2

(PM)

fraction over the microsome was detected in the absence and presence of Triton X-100, respectively. Taken together, the marker-enzyme and the NR activity data indicated that a membrane-associated NR activity was associated with the PM.

To determine whether NR activity was bound ionically to the PM, PM vesicles were resuspended in buffer containing 0- M NaCl, vigorously mixed for 30 s, sonicated on ice for min, and then pelleted. The control (no salt) fraction was not sonicated. The PM-associated NR activity was not affected by the salt washes up to 1 M. This indicated that the binding of NR activity to the PM was more than a simple ionic association and that NR activity was tightly associated with the lipid bilayer.

The Triton X-100 stimulation of membrane-associated NR activity (Tables 1, 3) indicates that the NADH oxidation sites of the PM NR activity and or N0 3 reducing sites were not accessible to NADH and or N0 3 NADH dependent ferricyanide reductase activity associated with corn root PM fractions was also stimulated six-fold by 0.025% Triton X-100, leading Buckhout and Hrubec (Buckhout, and Hrubec, T.C., Protoplasma 135:144-154 (1986)) to suggest that the primary site of NAD(P)H oxidation and or ferricyanide reduction was not exposed to the outside surface of the PM. Singer (Singer, Annu. Rev. Biochem. 43:805-833 (1974)) proposed that WO 91/04325 PCT/US90/04397 -43detergent activation was characteristic of enzymes which are deeply buried in a membrane.

For Table 3, the fractions were isolated as described above, and NR activity was determined in the presence and absence of 0.1% Triton X-100. The T:p ratio was appoximately 7. The experiment was repeated 5 times amd the means and SE are shown.

TABLE 3 Nitrate Reductase Activity in Corn Root Fractions Nitrate Reductase Activity Specific (nmol.mg protein.h- 1 Vol Protein Sample (ml) (mg/ml) -Triton +Triton 12 K Supernatanta 65 3 0.65 0.08 158 13 166 13 Solubleb 57 4 0.65 0.1 179 24 218 Microsomec 14 0.2 0.8 0.1 11 1 126 4 LI 2.4 0.5 0.8 0.1 3.8 0.5 90 12

U

2 1.4 0.2 0.8 0.05 19.3 0.5 323 8 a Post-12,000 g supernatant b Post-120,000 g supernatant c pellet from 120,000 g centrifugation LI first lower phase u 2 second lower phase To determine if the stimulation of NR activity by Triton X-100 were a result of solubilizing NR activity from the membrane, PM vesicles were treated with increasing concentrations of Triton X-100 and then centrifuged (Table 4).

Most of the NR activity was solubilized by 0.05% Triton X-100 (T:p 1.25), which is slightly less than the optimum T:p ratio for NR activity in microsome fractions (Table In contrast, little vanadate-sensitive H+-ATPase was removed by WO 91/04325 PCI/US90/04397 -44treatment of PM vesicles with up to 0.1% Triton X-100 (T:p (Table Unlike the PM ATPase it appeared that the latency of the PM-associated NR activity was a function of the solubilization of NR activity from the membrane.

The values for NR activity are higher in Table 4 than in Table 3 because the results are based on the protein concentration of the reaction mixture. In Table 3, the protein analysis included the membranes, whereas in Table 4, the membranes were separated from the preparation by centrifugation after NR activity was soluibilized by the Triton X-100 treatment. The concentration of protein in the supernatant solution was then determined.

For Table 4, twice pelleted and resuspended PM fractions were brought to 0 Triton X-100 for 15 min on ice and then centrifuged at 150,000 g for 15 min. Nitrate reductase (A and B) and vanadate sensitive ATPase activity (C and D) were determined in the soluble fractions (A and C) and the resuspended pellets (B and The experiment was repeated at least three times. Means values SE are shown.

WO 91/04325 PCT/US90/04397 TABLE 4 Triton X-100-solubilization of PM NR activity and H+-ATPase from corn roots Nitrate Triton T:p Protein reductase ac ivi y (mg.ml1) (nmol.mg protein- .h A. Soluble fractions 0 0 0 0 0 0.005 0.125 0.084 128 15.2 0.05 1.25 0.16 545 39 0.1 2.5 0.2 460 B. Membrane pellets 0 0 0.76 02 5.9 0.005 0.125 0.49 164 9.8 0.05 1.25 0.43 21 1 1.8 0.1 2.5 0.30 19 2.3 C. Soluble fractions 0 0 0 0 0 0.005 0.125 0.005 0 0 0.05 1.25 0.12 14 0.1 2.5 0.136 25 2.2 D. Membrane Pellets 0 0 0.27 23 2.1 0.005 0.125 0.20 34.4 3.1 0.05 1.25 0.16 75 13.9 0.1 2.5 0.092 120 14.2 EXAMPLE 4 CHARACTERISTICS OF MEMBRANE ASSOCIATED CORN NR ACTIVITY Plasma membrane NR activity was induced by 1 mM N0 3 above a very low control level which was present in PM WO 91/04325 PCT/US90/04397 -46fractions isolated from roots of plants rigorously kept free of NO 3 This low amount of NR activity may be constitutive or the result of a low level of NO 3 contamination which we are unable to detect in the system. Membrane NR activity was unstable; time dependent decreases in PM NR activity occurred when the PM fractions were kept at O'C in the presence of protease inhibitors. Plasma membrane NR activity solubilized with Triton X-100 and the soluble NR activity from corn roots were inactivated by antiserum prepared to NR activity purified from barley leaves. The anti-NR activity serum (Somers, D.A., et al., Plant Physiol. 72:949-952 (1983)) was kindly supplied to us by Dr. R. L. Warner, Washington State University, Pullman, WA.

In summary, latent NR activity was identified in corn-root PM fractions isolated by aqueous two-phase partitioning. The NR activity was not removed from PM vesicles by washing with increasing NaCl concentration up to 1 M but it was solubilized by Triton X-100 The results show that an integral membrane protein capable of reducing nitrate is present in the PM of corn roots.

EXAMPLE EFFECT OF ANTI-NR IgG ON THE ACTIVITY OF MEMBRANE ASSOCIATED BARLEY NR Anti-NR IgG fragments purified from Chlorella NR antiserum inhibited N0 3 uptake by more than 90% but did not affect N0 2 uptake by barley seedlings (Table (Chlorella NR was used as an antigen because it is more stable than barley NR and pure preparations were obtained (Hageman, R.H. et al., Enzymol. 69:270-280 (1980); Tischner, Planta 160:1-5 (1984)). The antiserum cross-reacted with soluble NR from barley roots as evidenced by inactivation of NR activity).

WO 91/04325 PCT/US90/04397 -47- The inhibitory effect was only partially reversible; uptake recovered to 50% of the control after 3 rinses in fresh uptake solution and a 10 min equilibration period. IgG fragments isolated from preimmune serum inhibited nitrate uptake 36% but the effect was completely reversible by rinsing.

Intact anti-NR molecules did not affect N0 3 uptake (Table presumably because they are much larger (150 kD) than the cleaved fragments (50 kD) and could not move as easily through the root cell wall to bind to N0 3 transport sites. Intact antibodies to the yeast PM Pi binding protein did not affect in vivo Pi uptake until the yeast cell wall was removed or the IgG molecules were cleaved with papain. This lead Jeanjean et al. (Jeanjean, R. et al., Arch. Microbiol. 137:215-219 (1984)) to suggest that the intact IgG molecules were too large to penetrate the yeast cell wall to bind to the Pi transporter.

The inhibition of N0 3 uptake by anti-NR IgG fragments suggested that NR or an antigenically related protein involved in N0 3 transport were present in the PM, the primary barrier of ions (and IgG fragments) to the cell cytosol. To determine if NR were present in the PM, it was important to isolate highly enriched PM fractions. Aqueous two-phase partitioning of plant membrane fractions is a convenient technique for isolating PM fractions of high purity (Hodges, T.K. et al., Meth. Enzvmol. 118:41-54 (1986); Larsson (Plasma Membranes, In: Modern Methods of Plant Analysis, New Series, Vol. 1, Cell Components, HF Linksens, et al., eds., Springer- Verlag, Berlin, pp. 85-104 (1985)).

For Table 5, uptake experiments were carried out with N0 3 induced (N0 3 uptake experiments) or N0 2 induced (N0 2 uptake experiments) as described above. Average rates SE for the 0.5 h uptake studies are reported. Each experiment was repeated 2 times and representative data are shown.

7 WO 91/04325 PCT/US90/04397 -48- TABLE Effect of NR Antibodies on Nitrate and Nitrate Uptake Expt Order Treatment N0 3 Uptake Rate NO0 2 Uptake Rate gmole/g x h 1. Initial uptake rate 3.3 0.1 3.6 0.4 2. Preimmune IgG Fragments 2.1 0.1 3.2 3. Preimmune IgG Fragments After Wash 3.0 0.09 3.1 0.4 4. Intact Anti-NR Serum 3.3 0.2 3.0 0.2 Intact Anti-NR Serum After Wash 3.69 0.1 3.3 0.4 6. Anti-NR IgG Fragments 0.25 0.08 3.2 0.9 7. Anti-NR IgG Fragments After Wash 1.89 0.1 2.9 0.2 EXAMPLE 6 PLASMA MEMBRANE ASSOCIATED NITRATE REDUCTASE ACTIVITY IN BARLEY Marker enzyme assessment of two-phase partitioned barley root microsomal fractions is shown in Table 6. A 2.3 to 2.8 fold enrichment of the PM ATPase activity was detected in the upper phase over the microsome fraction in the presence and absence of Triton X-100, respectively. The Triton stimulation of the ATPase of phase partitioned PM fractions is well established (Table refs. Larsson (Plasma Membranes, In: Modern Methods of Plant Analysis, New Series, Vol. 1, Cell Components, HF Linksens, et al., eds., Springer-Verlag, Berlin, pp. 85-104 (1985)); Sandstrom, R.P. et al., Plant Phvsiol. 85:693-698 (1987)). The latency indicates that the vesicles are sealed and right side out and is attributed to increased accessibility of ATP to the hydrolytic site of the PM ATPase. Recently, it has been proposed that Triton X-100 WO 91/04325 PCT/US90/04397 -49also activates the PM ATPase possibly by altering the lipid environment near the ATPase possibly by altering the lipid environment near the ATPase or by removing an inhibitory component (Sandstrom, R.P. et al., Plant Physiol. 5:693-698 (1987)). Evaluation of markers for the tonoplast, Golgi apparatus, mitochondria, ER, plastid and cytoplasm demonstrated that each of these activities was significantly reduced by the two-phase procedure. Of particular significance was the absence of cytoplasmic contamination in the upper phase fractions since NR is generally considered to be a cytosolic enzyme (Oaks, A. et al., Ann. Rev. PI Physiol.

36:345-365 (1985)). The marker enzyme data indicate that the upper phase fraction consisted mainly of PM.

In Table 6, root fractions were isolated and enzymes were assayed as described above. Vanadate sensitive ATPase in the presence and absence T) of 0.0125 Triton X-100 (T:P N0 3 sensitive ATPase and latent UDPase activities are in Amol Pi/ mg protein x h. NADH Cyt c reductase and Cyt c oxidase activities are in p~ole/mg protein x min. TPI and ADH activities are in nmole NADH oxidized or pmole NAD reduced per mg protein x min, respectively. Each experiment was repeated at least 3 times and representative data are shown.

WO 91/04325 PCT/US90/04397 TABLE 6 Marker Enzyme Assessment of Soluble, Microsome and Phase Partitioned Membrane Fractions Marker Plant Phase Fraction Soluble Microsome Lower Upper Vanadate sensitive ATPase PM 11.8 22.1 21.2 51.8 Vanadate sensitive ATPase PM 14.3 30.3 24.5 84.5 NO" sensitive ATPase tonoplast 0.70 0.31 0.95 0 Latent UDPase Golgi 3.2 4 10.4 0 NADH Cyt c reductase ER 66 203 195 55.2 Cyt c oxidase mitochondria 0 37.9 97.5 0.64 TPI plastid/cytoplasm 0.27 2.44 2 0 ADH -vtoplasm 50.1 0 0 0 EXAMPLE 7 DISTRIBUTION OF BARLEY NITRATE REDUCTASE ACTIVITY The NR activity distribution in root soluble and phase partitioned membrane fractions is shown in Table 7. Most of the NR activity was soluble as is well established (Oaks, A.

et al., Ann. Rev. PI Physiol. 36:345-365 (1985)); however, approximately 4% of the total NR was associated with the membrane fraction when assayed in the presence of Triton. A WO 91/04325 PCT/US90/04397 -51membrane fraction when assayed in the presence of Triton. A 1.8 to 5.4 fold enrichment of the NR activity in the upper phase (PM) fraction over the microsome in the presence and absence of Triton, respectively, (Table 7) was detected.

Repeated resuspension and repelleting of the PM fractions failed to decrease NR activity. Taken together, the results from the marker enzymes and from the NR activity show that a significant portion of barley root NR was present in highly enriched PM fractions.

The addition of 0.1% Triton X-100 (T:P 10) to the reaction mixture stimulated membrane associated NR specific activity 60 fold in the microsome fraction, 16 fold in the lower and 20 fold in the upper phase, but increased soluble NR activity by only a factor of 1.7 (Table In contrast to NR, upper phase PM ATPase activity was stimulated by Triton whereas lower phase PM ATPase activity was not affected (Table These results agree with others who found that inside-out PM vesicles partition in the lower phase of aqueous polymer two-phase systems (Larsson Plasma Membranes, In: Modern Methods of Plant Analysis, New Series, Vol. 1, Cell Components, HF Linksens, et al., eds., Springer-Verlag, Berlin, pp. 85-104 (1985)). Triton X-100 does not stimulate the PM ATPase of lower phase vesicles because of hydrolytic site of the enzyme is on the outside and is accessible to ATP (Larsson Plasma Membranes, In: Modern Methods of Plant Analysis, New Series, Vol. 1, Cell Components, HF Linksens, et al., eds., Springer-Verlag, Berlin, pp. 85-104 (1985); Sandstrom, R.P. et al., Plant Physiol. 85:693-698 (1987)).

The lack of significant NR activity in the absence of Triton in both the lower and upper phase PM fractions indicated that the membrane associate NR N03" reducing and/or NADH oxidizing sites were not directly exposed to either side of the PM.

Most of the PM associated NR was solubilized by 0.1% Triton X- 100 (T:P 2.5) (Table In contraiL, similar WO 91/04325 PCT/US90/04397 -52concentrations of Triton did not solubilize the PM ATPase.

This suggests that the latency of the membrane associated NR was a function of the solubilization of NR from the membrane.

For Table 7, root fractions were isolated as described above. Nitrate reductase was assayed in the presence and absence of 0.1% Triton X-100 (T:P 10). The experiment was repeated at least 4 times and data from a representative experiment are shown.

For Table 8, upper phase (PM) fractions were isolated and treated with Triton X-100 as described above. NR was assayed in the soluble and pellet fractions in the presence of 0.1% Triton X-100. The experiment was repeated 5 times data from a representative experiment are shown.

c' _L WO 91/04325 PCT/US90/04397 -53- TABLE 7 Nitrate Reductase Activity Distribution in Barley Root Fractions Nitrate Reductase Activity Total Specific Sample Vol Prot -Triton +Triton -Triton +Triton (ml) (mg/ml) (nmol/h) (nmol/gm prot. x h) 12K Supernatanta 65 0.56 10,300 21,300 284 586 Solubleb 59 0.5 11,600 19,200 394 651 ticrosomec 13 0.41 13.9 840 2.6 157 Lower Phase 1 2.2 0.48 7.1 116 6.7 110 Upper Phase II 1.1 0.35 5.4 109 14 284 afrom 12,000 g centrifugation bfrom 120,000 g centrifugation Cpellet from 120,000 g centrifugation TABLE 8 Triton Solubilization of PM Associated Barley NR Treatment Nitrate Reductase Activity Pellet Soluble nmole/mg protein x h -Triton X-100 211 0 +Triton X-100 26 301 I I

-A

wih; WO 91/04325 PCT/US90/043Q7 -54- EXAMPLE 8 EXTENT OF THE ASSOCIATION OF BARLEY NITRATE REDUCTASE AND THE PLASMA MEMBRANE To determine the extent of the association of NR and the PM, PM vesicles were vigorously vortexed in 500 mM NaC1 and 1 mM EDTA, sonicated for 15 min and then pelleted after dilution (Table The salt/chelate/sonication treatment did not remove NR from the PM fraction suggesting that the association was not simply ionic but that NR was tightly associated with the PM lipid bilayer.

To further characterize the PM associated NR activity, PM vesicles were isolated, treated with Triton X-100 (T:P to remove NR from the membrane pellet (Table 8) and then incubated with either preimmune or anti-NR serum (Table The PM associated NR was inactivated by NR antiserum but was not affected by the preimmune serum. A single spot on Western blots of Triton solubilized PM fractions separated by twod-mensional electrophoresis was detected with the NR antiserum. preparation).

The inactivation of the PM associated NR and the inhibition of N0 3 transport by the NR antiserum present the possibility that N0 3 transport and the PM associated NR may be related. The PM associated NR may be a part of an enzyme complex that both transports and reduces N0 3 as proposed by ,Butz and Jackson (Rutz, R.G. et al., Phytochem. 16:409-417 (1977)). Alternatively, the N0 3 transporter and the PM associated NR may be completely separate but antigenically related systems. Rufty et al. (Rufty, Jr. et al., Plant Phvsiol. 82:675-680 (1986)) recently propose the possibility that the receptor for the NR induction system and the

I

WO 91/04325 PCT/US90/04397 functional NR protein may both be associated with the PM of root cells.

The results demonstrated that anti-NR IgG fragments specifically inhibited N0 3 uptake. As shown herein, localized membrane associated NR in the PM ashown that the same antibodies that inhibited N0 3 uptake also inactivated PM NR. These results indicate a possible relationship between

NO

3 transport and N0 3 reduction in the PM of barley roots.

For Table 9, plasma membrane fractions were isolated and washed with or without 500 mM NaCl, 1 mM EDTA and sonication as described above. After treatment, the PM fractions were diluted with resuspension buffer and pelleted. The resulting pellets were assayed for NR activity in the presence of Triton X-100 (T:p 10). The experiment was repeated 3 times and data from a representative experiment are shown.

For Table 10, nitrate reductase was solubilize from the PM fractions as described above. One hundred il of water antiserum), preimmune serum, or anti-NR antiserum was added to 200 pl of the Triton solubilized PM fractions. The fractions were kept on ice for 2 h and then centrifuged for 5 min at 12,000 RPM in a microfuge. Nitrate reductase was assayed in the supernatant fractions. Each experiment was repeated at least 4 times and representative date are shown.

F

WO 91/04325 PCT/US90/04397 -56- TABLE 9 Salt and Chelate Wash of Barley PM Fractions Treatment Nitrate Reductase Activity (nmole/mg protein x h) -NaCI 211 +NaC1, EDTA 224 TABLE Inactivation of Barley PM NR Solubilized with Triton X-100 Treatment Nitrate Reductase Activity (nmole/ml x h) -Antiserum +Preimmune serum Anti-NR serum 15.8 16.0 0.0 While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

Claims (11)

1. A plasma membrane associated nitrate reductase enzyme activity, said enzyme activity being substantially free of natural contaminants, wherein said enzyme activity is derivable from plants.

2. The plasma membrane associated nitrate reductase enzyme activity of claim 1 wherein said enzyme activity is derivable from corn.

3. The plasma membrane associated nitrate reductase enzyme activity of claim 1 wherein said enzyme activity is derivable from barley.

4. A recombinant molecule containing a gene sequence capable of encoding a plasma membrane associated nitrate reductase enzyme activity, wherein said enzyme activity is derivable from plants.

The recombinant molecule of claim 4 which expresses said plasma membrane associated nitrate reductase enzyme activity.

6. The recombinant molecule of claim 5 wherein said molecule is capable of expressing said enzyme activity in a bacterial cell.

7. The recombinant molecule of claim 5 wherein said molecule is capable of expressing said enzyme activity in a plant cell.

8. A cell containing the recombinant molecule of claim 4.

9. The cell of claim 8 which is a bacterial cell.

The cell of claim 8 which is a plant cell. 7III' -58-

11. A plant containing a recombinant molecule containing a gene sequence capable of encoding a plasma membrane associated nitrate reductase enzyme activity. Dated this 8th day of April, 1993. THE GENERAL HOSPITAL CORPORATION and THE REGENTS OF THE UNIVERSITY OF CALIFORNIA Patent Attorneys for the Applicant: PETER MAXWELL ASSOCIATES 0 0 R~ *1 INTERNATIONAL SEARCH REPORT International Application No PCT/US90/04397 I. CLASSIFICATION OF SUBJECT MATTER (if several classification symbols apply, Indicate all) 3 I I I I I I According to International Patent Classification (IPC) or to both National Classification and IPC C12N 09/02; C12N 09/06; C12N 15/00; A01H 05/00 U.S. Cl: 435/189; 435/191;435/172.3; 800/205 II. FIELDS SEARCHED Minimum Documentation Searched Classification System Classification Symbols U.S. Cl.: 435/189; 435/191; 435/172.3; 800/205; Documentation Searched other than Minimum Documentation to the Extent that such Documents are Included in the Fields Searched 6 WORLD PATENT INDEX; DIALOG: BIOTECH; CHEMICAL ABSTRACTS III. DOCUMENTS CONSIDERED TO BE RELEVANT 1 Category Citation of Document, i1 with indication, where appropriate, of the relevant passages t Relevant to Claim No. it x The EMBO Journal, Volume 7, no. 11, 1-11 issued 1988, C-L Cheng et al., "A New Locus (NIAI) in Arabidopsis thaliana encoding Nitrate Reductase," see pages

3309-3314. X NATO Advanced Science Institute 1-11 Series: Series A, Volume 61, Genetic Engineering in Eucaryotes, issued 1983, A. Kleinhofs et al., "Nitrate Reductase Genes as Selectable Markers for Plant Cell Transformation", see pages 215- 231. N FEMS Microbiology Letters, Volume 11, 1-6, 8-q y issued 1981, C.G. Barr et. al., "Cloning 7, 10, 11 the chl C Gene for Nitrate Reductase of Escherichia coli pages 213-216,see entire document. Special categories of cited documents: I later document published after the internationai filing date A" document defining the general state of the art which is not or priority date and not in conflict with the application but A" dcumentd d efnn h ar e ticular releancthe whch not cited to understand the principle or theory underlying the considered to be parcular relevance invention earlier document but published on or after the international document of particular relevance: the claimed Invention filng date cannot be ccnsidered novel or cannot be considered to document which may throw doubts on priority claim() or involve an inventive stop which is cited to estabish the publication date of another document of particular relevance; the claimed invention citation or other special reason (as specified) cannot be considered to involve an inventive step when the document referring to an oral disclosure, use, exhibition or document is combined with one or more other such docu- other means ments, such combination being obvious to a person skilled document published prior to the international filing date but in the art. later than the priority date claimed document member of the same patent family IV. CERTIFICATION Date of the Actual Completion of the International Search 2 Date of MaIling of this International Search Reoort 1 19 November 1990 JAN 1991 International Searching Authority I .Snature of Authorlzed Officer ISA/US _Marian C, Knode Form PCT/ISA/210 (second sheet) (May 1986) International Application No. PCT/US90/04397 FURTHER INFORMATION CONTINUED FROM THE SECOND SHEET Gene, Volume 85, no. 2, issued 1989, F. Daniel-Vedele, "Cloning and Analysis of the Tomato Nitrate Reductase-Encoding Gene: Protein Domain Structure and Amino Acid Homologies in Higher Plants", see pages 371-380. 1-9 10-11 V OBSERVATIONS WHERE CERTAIN CLAIMS WERE FOUND UNSEARCHABLE' This international search report has not been established in respect of certain claims under Article 17(2) for the following reasons: 1 Claim numbers .because they relate to sublect matter I not required to be searched by this Authority namely: 2. Claim numbers ,because they relate to parts of the international application that do not comply with the prescribed require- ments to such an extent that no meaningful international search can be carried out i, specifically: 3. Claim numbers because they are dependent claims not drafted in accordance with the second and third sentences of PCT Rule 6.4(a,. VI OBSERVATIONS WHERE UNITY OF INVENTION IS LACKING' This International Searching Authority found multiple inventions in this international application as follows: See Attachment. As all required additional search lees were timely paid by the applicant, this international search report covers aii searchable claims of the international application. Telephone practice As only some of the required additional search fees were timely paid by the applicant, this international seoi report covers only those claims of the International application for which fees were paid, specifically claims: 3.M No required additional search fees were timely paid by the applicant. Consequently, this international search report is testricted to the invention first mentioned in the claims; !t is covered by claim numbers: 4 As all seaichable claims could be searched without effort lustiying an additional lee, the International Searching Authority did not invite payment of any additional fee. Remark on Protest The additional search tees were accompanied by applicant's protest. E No protest accompanied the payment of additional search fees. Form PCT/ISA/21O (supplomontal shoot (Rev. 4.90) International Application No. P' CT US 9 0 04 39 7 Ill. DOCUMENTS CONSIDERED TO BE RELEVANT (CONTINUED FROM THE SECOND SHEET) Category I Citation of Document, with indication, where appropriate, of the relevant passages 17 Relevant to claim No Biochemica et Biophysica Acta, 11-.1 V \olumie 708, issued 1982, T.M. Kuo 4-11 1et. al., "NADH-Nitrate Reductase in Barley Leaves identification *and Amino Acid Composition of Sub Unit Protein", see pages 75-81. X Eur. J. Biochemistry,- Volume 158, 1 issued 1986, Craske et. al., "The *Respiratory Nitrate Reductase from Paracoccus denitrifica-Xs," see page 429-436. JP;A4.,132382 GEN Hospital Corp) 24 1-11 >Iav 1989, see whole document. Form PCT/ISA/210 (extra sheet) (May 1986) PCT/US9O/04397 Attachment To Form PCT/ISA/210 Attachment to Part VI, OBSERVATIONS WHERE UNITY OF INVENTION IS LACK C2 ~1-1 cdravn a nitt aterdits acriv tv, as, fle in ClIass 47,9, subclasse.3 130 avid 19,fkr e,-aip'. T T Cla ams 6 a nd 83-q, d ra vtn a rec'nb -a nr woI tle,, X teS'; ig i1t1a t e e du C't asef and bact e r ialI, re IIs ta in in a (niih i ia n r moeI c uIe s e \p ress in g n i rat P r edu 11 r5 r. t P a H~si i e class subclass 172.3', for eNamfple.. TTT Cl a 4Ls 7 -38, a nd 10 -l11, d ra wn toc a re(.cnmb in an t nh1 ecul- c-,-.:pressi ug nitrate reductase and plants and plant ce!Ls1 ~nt~ nncrmbinint molecules e~pressing nitratte reduc ras;( lsificed in Class 800, subri ass 205, fcr (-Nanple. The nvr *nsare li st n-t, zach f rn the, otrhr becallse 2i l g esoi ,-ntinsTT and T are rel a td as miutual c1iie p- -,ts In iterfled(iate-final product relat!ionship. Dtnt~ s Drk~v-nr f,.r -lairis in this rplarJL-nship if rte iirermprlii s usefiul fn make other than the f inal p-ro~dtwt .aid thp Tn rhe -instant -aqe, the intermediate product 'Is de(-wt'-d to) bre M.f as a h\vbricdi at ion probep antd the nvn ti ar ~atltal~distinct since there is nothing ttn this rtr to s o0 themi t,)tn be bv4,is variants. Tn\ entions TIT and I are related as mutuial ly s ~in nternicdiati-final producit relationship. 04 sr, nctrness en f,-r ,-1aims in this relationship if the 'interniediatte I>I r-useful to make other than the f inal product :adw rth es ar pat.eritabl\ distinct. Tn the instant -asp t he n termediar-w- product s ,Heen1Hkd] he useful as a hvbri diza tion prob)Ie and the- in rv- ntions aip Iftm- pa ten tably distinct since( thero is it t hi ng t n t hi s rta Jut *hpm to be obvious variants. The invt-ntirns rvoiips IT and ITT are independlent and d3is;tinc7t-hbecause e-.pression of a recombinant DN mioltecult- ini a ha('tferiAl l requires different consideraticons and '.ou)ldr have a -;eparare in~e than e, prossion in a plant olr pl ant ce2 2.