WO1998005758A1 - STRUCTURE AND EXPRESSION OF THE ALPHA-CARBOXYLTRANSFERASE SUBUNIT OF HETEROMERIC-ACETYL-CoA CARBOXYLASE - Google Patents

Abstract

The nucleic acid sequence encoding the alpha-carboxyltransferase (alpha-CT) subunit of acetyl-CoA carboxylase and its deduced amino acid sequence are provided. Vectors comprising the nucleic acid sequence, plant cells transformed with the vectors, as well as plants transformed with the nucleic acid sequence and seeds of the transgenic plant, are also provided. By controlling the expression of the alpha-CT gene, carboxylation of acetyl-CoA can be controlled. Thus, by introducing constructs of the gene of the present invention in sense or antisense orientation, carboxylation of acetyl-CoA to produce malonyl-CoA may be increased or decreased and fatty acid synthesis and elongation in plants and seeds which depend on malonyl-CoA may be controlled. The figure shows ACCase and CT activities and protein profiles.

Description

STRUCTURE AND EXPRESSION OF THE

ALPHA-CARBOXY TRANSFERASE

SUBUNIT OF HETERO ERIC-ACETYL-CoA CARBOXYLASE

SPONSORSHIP

Work on this invention was sponsored in part by National Science Foundation Grant DCB 90-05290. The Government may have certain rights in the invention.

FIELD OF THE INVENTION The present invention relates generally to the σ-carboxyltransferase subunit of acetyl-CoA carboxylase and more particularly, the σ-carboxyltransferase subunit of heteromeπc acetyl-CoA carboxylase from pea (Pisυm sativυm L.) and its use in controlling the carboxylation of acetyl-CoA in plants

BACKGROUND OF THE INVENTION Acetyl-CoA carboxylase (ACCase) catalyzes the first step in fatty acid biosynthesis leading to the synthesis of malonyl-CoA from acetyl-CoA. Examination of this reaction in vitro and in vivo has implicated it as a key regulatory step for plastidial fatty acid biosynthesis in spinach (Post-Beittenmilier, D., et al. J. Biol. Chem. 266.1858-1865 (1991) and Post-Beittenmiller, D , et al Plant Physiol. 100.923-930 (1992)), barley and maize leaves (Page, R A , et al Biochem Biophys Acta. 1210 369-372 (1994)), wheat (Eastwell, K C, et al Plant Physiol. 72.50-55 (1983)) and tobacco suspension cells (Shintani, D K , et al , The Plant J. 7:577-586 (1995))

Because of its pivotal role in fatty acid synthesis and as a target for herbicides (Burton, J.D , et al Pestic. Biochem Physiol 34 76-85 (1989)), much work has been focused on characteπzing the structure of plant ACCase. This research has recently led to the finding of two isozymes of ACCase localized in different subcellular compartments. Konishi, T., et al Proc Nat Acad. Sci. USA, 91 :3598-3601 (1994) and Sasaki, Y., et al., Plant Physiol 108:445-449 (1995) While malonyl-CoA produced in the plastids is used predominantly for the synthesis of fatty acids, cytosolic malonyl-CoA is used for the biosynthesis of very long chain fatty acids (Pollard M, et al., Umnathes alba Plant Physiol. 66:649-655 (1980)) and flavonoids (Ebel, J., et al., Petroselinum bortense. Eυr. J. Biochem. 75:201-209 (1977) and Ebel, J., et al., Arch. Biochem. Biophys. 232.240-248 (1984)), as well as for the malonation of ammo acids or 1-amιnocyclopropane-1-carboxylic acid. The presumably cytosolic form of plant ACCase is similar to mammalian ACCase (Lopez- Casiilas, F et al., Pros. Natl Acad. Sci. USA, 85:5784-5788 (1988); Takai, T., et al., J. Biol. Chem. 263:2651-2657 (1988); and Ha, J., et al., Eur. J. Biochem. 219:297- 306 (1994)) in that it is a multi-functional polypeptide referred to as the homomeric form. Cytosolic ACCase has a molecular mass of more than 200 kd and contains the biotin carboxyl carrier protein (BCCP), biotin carboxylase (BC), and β- carboxyltransferase (S-CT) subunits as functional domains (Egin-Buhler, B., et al., Eur. J. Biochem. 133:335-339 (1983); Egli, .A., et al., Plant Physiol. 101 :499-506 (1993); Slabas, A.R., et al., Plant Sci. 39:177-182 (1985); Charles, D.J., et al., Phytochemistry 25:1067-1071 (1986); Bettey, M., et al, J. Plant. Physiol. 140: 513- 520 (1992); Egli, M.A., et al., Plant Physiol. 101 :499-505 (1993); Gornicki, P., et al., Plant Mol. Biol. 22:547-552 (1993); Roessler, P.B., et al., Cyclotella cryptica. J. Biol. Chem. 268:19254-19259 (1993); Shorrosh, B.S., et al., Proc. NatI Acad. Sci. USA, 91 :4323-4327 (1994); Gornicki, P., et al., Proc. NatI Acad Sci. USA, 91 :6860-6864 (1994); Roesler, K.R., et al., Plant Physiol. 105:61 1-17 (1994); Ashton, AR. et al., Plant Mol. Biol. 24:35-49 (1994); Elborough, K.M., et al , Biochemical J. 301 :599- 605 (1994); and Schulte, W., et al., Plant Physiol 106 793-794 (1994).

The plastidial form of ACCase in most plants is similar to prokaryotic ACCase (Alix, J-H. DNA, 8:779-789 (1989); Kondo, H , et al , Proc. NatI Acad. Sci. USA, 88:9730-9733 (1991); Li, S-J., et al., J. Biol Chem 267 855-863 (1992); and Li, S-J., et al., J. Biol. Chem. 267:16841-16847 (1992)) tn that it is a heteromeric enzyme composed of dissociable subunits of different sizes, and it is thus referred to as the multi-subunit (MS) form. Dehaya, L., et al , Eur J Biochem. 225:1113- 1123 (1994); Alban, C, et al. Plant Physiol. 109:927-935 (1995) To date, the BCCP (Choi, J-K., et al., Plant Physiol. 109:619-625 (1995)), β-CT (Sasaki., Y„ et al., J. Biol. Chem. 268:25118-255123 (1993)), and BC (Shorrosh, B.S., et al., Plant Physiol. 108:805-812 (1995)) subunits of the chloroplast-heteromeric ACCase have been characterized and cloned, and these three subunits share substantial sequence similarity with bacterial ACCase subunits. However, carboxyltransferase (CT) activity has not been purified, and the σ-CT subunit has not been identified. Progress has been hindered by the tendency of the plastidial ACCase to lose activity upon purification and by the lack of a convenient assay for CT

It would thus be desirable to provide the σ-CT subunit of acetyl-CoA carboxylase. It would also be desirable to control expression of the gene encoding the σ-CT subunit of acetyl-CoA carboxylase. It would further be desirable to control the carboxylation of acetyl-CoA to produce malonyl-CoA It would also be desirable to control the carboxylation of acetyl-CoA to produce malonyl-CoA by controlling the expression of a gene encoding an ACCase subunit. It would further be desirable to acquire long-term control of the carboxylation of acetyl-CoA to produce malonyl-CoA by genetically altering plants. It would also be desirable to control fatty acid synthesis in plants and seeds by controlling the carboxylation of acetyl-CoA to produce malonyl-CoA. It would further be desirable to control fatty acid synthesis in plants and seeds without employing foreign chemicals.

SUMMARY OF THE INVENTION A purified and isolated nucleic acid sequence encoding the σ- carboxyltransferase (σ-CT) subunit of heteromeric ACCase is provided. Vectors comprising the nucleic acid sequence, plant cells transformed with the vectors, as well as plants transformed with the nucleic acid sequence and seeds of the transgenic plants, are also provided. The σ-CT nucleic acid sequence may be used to control carboxylation of acetyl-CoA to produce malonyl-CoA. Thus, by introducing constructs of the gene in sense or anti-sense orientation, carboxylation of acetyl-CoA to produce malonyl-CoA may be increased or decreased. Consequently, fatty acid synthesis and elongation in plants and seeds which is dependent on malonyl-CoA may also be increased or decreased. Secondary metabolite production in plants which is also dependent on acetyl-CoA and malonyl-CoA may also be controlled. Moreover, long-term control of the carboxylation of acetyl-CoA to produce malonyl-CoA may be obtained by genetically altering plants with the σ-CT nucleotide sequence.

To assist in characterizing the σ-CT subunit of heteromeric ACCase, an improved assay was developed to follow CT purification. It was found that a previously sequenced pea chloroplast cDNA of unknown function (IEP96), with a predicted molecular weight of 91 kd, encodes the σ-CT subunit of the heteromeric ACCase. Although IEP96 had sequence similarity to the σ-CT subunit of E. coli ACCase, because there are numerous carboxylation reactions in plants which are catalyzed by proteins with related sequences, IEP96 may have represented a subunit of any number of enzymes.

Additional objects, advantages, and features of the present invention will become apparent from the following description taken in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

The various advantages of the present invention will become apparent to one skilled in the art by reading the following specification and by referencing the following drawings in which:

Figure 1 sets forth the results of the fractionation of pea chloroplast proteins by gel permeation chromatography:

Figure 1A shows ACCase and CT activities and protein profiles; Figure 1 B shows the determination of CT activity;

Figure 1C is a Western blot analysis of gel permeation fractions using BC, biotin (BCCP), ?-CT, and σ-CT antibodies;

Figures 2A, 2B and 2C show the immunoprecipitation of pea chloroplast ACCase and CT activities by ?-CT and σ-CT antibodies;

Figures 3A and 3B show DEAE analysis of gel permeation purified chloroplast ACCase;

Figure 4 shows two dimensional analysis of gel permeation purified chloroplast ACCase; and Figure 5 is a photograph of the Western blot analysis of pea leaf proteins.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS It has been shown that a nucleic acid sequence IEP96, with a previously unknown function, encodes the σ-CT subunit of chloroplast heteromeric ACCase. The cDNA and deduced amino acid sequence of IEP96 are set forth in SEQ ID Nos. 1 and 2, respectively. Sequences of the present invention may thus be used to increase or decrease the carboxylation of acetyl-CoA to produce malonyl-CoA in the plastid of plants, thereby increasing or decreasing fatty acid synthesis. A method of controlling carboxylation of acetyl-CoA to produce malonyl-CoA and controlling fatty acid synthesis is thus provided by the present invention. Vectors containing the σ-CT nucleic acid sequence, plant cells transformed with the vectors of the present invention as well as plants containing the sequences of the present invention and seeds produced thereby, are also within the scope of the present invention.

The methods of the present invention generally comprise the step of introducing in sense or antisense orientation the σ-CT gene described herein into a plant cell and growing the cell into a plant. It will be appreciated that other genes (both sense and antisense orientation) may also be introduced into the plant cell, e.g., genes encoding the other known subunits of the heteromeric form of ACCase, BCCP (Choi, J-K., et al., Plant Physiol. 109:619-625 (1995)), 0-CT (Sasaki., Y., et al., J. Biol. Chem. 268:25118-255123 (1993)), and BC (Shorrosh, B.S., et al., Plant Physiol. 108:805-812 (1995)).

The σ-CT gene in sense or antisense orientation may be fused to a gene or fragment thereof which allows the σ-CT gene to be transported and expressed in a plant cell. The σ-CT gene in sense or anti-sense orientation in combination with the gene or gene fragment is referred to as a "construct" herein. It will be appreciated that the constructs of the present invention may contain any regulatory elements necessary and known to those skilled in the art for expression of the σ-CT gene in either orientation. For example, constructs prepared with either seed-specific promoters such as the napin seed storage protein promoter of rapeseed, or with a constitutive promoter such as the cauliflower mosaic virus 35 S (CaMV35S) promoter, are contemplated by the present invention. It is believed that seed-specific promoters may be more desirable and effective in altering seed oil amounts or composition by avoiding possible deleterious effects in the plant. The constitutive promoter, however, may be more effective in, for example, engineering general herbicide resistance in the whole plant. Plants of the Gramineae family are extremely sensitive to certain "grass-selective" herbicides. This sensitivity is known to result from the inhibition of the homomeric ACCase. Certain grass biotypes which are partially resistant to the herbicides have been shown to have altered ACCase. The heteromeric ACCase is completely resistant to the "grass-selective" herbicides. Therefore, introduction of the gene of the present invention into Gramineae plants such as Gramineae grasses (e.g., corn, wheat, barley, oats, etc.), can provide resistance in these species to such herbicides. It will be appreciated that genes encoding the other subunits of the heteromeric ACCase may also be introduced in addition to or in combination with the gene of the present invention. Because herbicides are usually applied as foliar or ground applications it is necessary for the vegetative parts of the plant to be resistant. Therefore, a constitutive promoter, such as CaMV35S would be used in the construct to create such transgenic plants.

Because malonyl-CoA is required for fatty acid synthesis and elongation in plants and seeds, the present invention also provides a method of controlling plant and seed fatty acid synthesis and elongation. Increasing seed fatty acid synthesis by overexpressing the σ-CT gene is useful in increasing oil content of rapeseed, soybean, or other oilseed crops. In Brassica seeds, overexpression of the homomeric ACCase increases the malonyl-CoA/acetyl-CoA ratio and increases the amount of oil stored in the seeds. Because the homomeric ACCase is a 250 kDa protein, achieving high levels of expression and successfully targeting to the plastid at sufficiently high expression levels, may be more difficult than the overexpression of heteromeric ACCase. Increasing oil content of oil seeds by overexpressing the σ-CT gene is therefore advantageous. In addition, because the heteromeric ACCase gene described herein encodes a plastid protein and, since fatty acid synthesis takes place primarily in the plastid, a construct which includes the gene described herein does not require a plant plastid transit peptide. An effective increase in ACCase activity in the plastid thus results when the plastid ACCase gene of the present invention is overexpressed. It should be appreciated that decreasing seed fatty acid synthesis by decreasing gene expression is also useful in producing "low-fat" seeds such as low-fat peanuts.

As previously discussed, acetyl-CoA and malonyl-CoA are precursors of various plant secondary metabolites. Decreasing expression of σ-CT may therefore decrease fatty acid synthesis in the plastid and subsequent long chain fatty acid synthesis in the cytosol This increases the amount of cytosolic malonyl-CoA available for synthesis offlavonoids, isoflavonoids, and other secondary metabolites Furthermore, decreasing expression of the σ-CT gene set forth herein may ultimately decrease the amount of malonyl-CoA present and increase the amount of acetyl-CoA present Thus, altering expression of the σ-CT gene may favorably alter the amount of acetyl-CoA or malonyl-CoA available for production of secondary plant products, many of which have value in plant protection against pathogens or for medicinal or other uses Furthermore, it is not necessary that these products be naturally present in plants. For example, bacterial genes may be introduced into plants to produce polyhydroxybutyrate which can be used to synthesize biodegradable plastics Poirier Y et al , Science 256 520-524 (1992) Production of polyhydroxybutyrate or other acetyl-CoA derived products in a plant will require adequate supply of cellular and plastidial acetyl-CoA If the fatty acid synthesis pathway is drawing on this acetyl-CoA supply for oil storage, the amount available for alternative, higher-value products will be less Therefore, inhibition of the fatty acid synthesis pathway may be desirable to allow diversion of more carbon into products other than fatty acids, e.g., increasing the acetyl-CoA to malonyl-CoA ratio by decreasing ACCase gene expression may allow more carbon flux into polyhydroxybutyrate production thereby resulting in higher yields of polyhydroxybutyrate or other acetyl-CoA derived products

It will be appreciated that the methods of the present invention further include introducing the constructs of the present invention including the sense or antisense orientation of the σ-CT gene into a plant cell, and growing the cell into a callus and then into a plant; or, alternatively, breeding a transgenic plant produced from the above method with a second plant to form an F1 or higher hybrid (e.g., F2) For example, constructs containing the nucleotide sequences of the present invention may be introduced into plants by cocultivation with Agrobactenum containing the construct Transgenic plants are therefore produced by the methods of the present invention and are also contemplated by the present invention . γ _

As referred to herein, the term "gene" is meant a nucleic acid, either genomic or synthetic, which encodes a protein product. The term "nucleic acid" is intended to mean natural and synthetic linear and sequential arrays of nucleotides and nucleosides, e.g., cDNA, genomic DNA (gDNA), mRNA, and RNA, oligonucleotides, oligonucleosides, and derivatives thereof. Thus, alternate nucleic acid forms such as genomic DNA, cDNA and DNA prepared by partial or total chemical synthesis from nucleotides, as well as DNA with mutations, are also within the contemplation of the invention.

The term "sense orientation" as used herein refers to the orientation of a gene such that its RNA transcript, following removal of introns, is translatable into the polypeptide product of the gene. The term "antisense orientation" is used to mean the opposite orientation of a gene such that its transcript is complementary to the normal transcript of the gene when in sense orientation. In addition, the term "encoding" is intended to mean that the subject nucleic acid may be transcribed and translated into either the desired polypeptide or the subject protein in an appropriate expression system, e.g., when the subject nucleic acid is linked to appropriate control sequences such as promoters, operators, regulators, and the like, in a suitable vector (e.g., an expression vector) and when the vector is introduced into an appropriate system or cell. By "substantially represented by" or "substantially complementary to" as used herein is meant any variation therein which does not impair the functionability of the sequence to any significant degree. By "substantially as shown" or "substantially similar" with respect to a nucleic acid is meant sufficiently similar in structure or sequence to encode the desired polypeptide or gene product, or with respect to a polypeptide, sufficiently similar in structure or sequence to serve its principal function. It will thus be appreciated that the term "polypeptides" includes not only full length protein molecules but also fragments thereof which, by themselves or with other fragments, generate substantially similar physiological activity as the full length protein. It will further be appreciated that synthetic polypeptides of the protein of the present invention are also within the scope of the invention and can be manufactured according to standard synthetic methods.

The terms "oilseed plant" and "oilseed crop" are used interchangeably herein and refer to those plants and crops known to those skilled in the art as part of the oilseed variety, including but not limited to rapeseed, soybean, Crambe, mustard, castor bean, peanut, sesame, cottonseed, linseed and sunflower. The term "capable of hybridizing under stringent conditions" is used to mean annealing a first nucleic acid to a second nucleic acid under stringent conditions (defined below). For example, the first nucleic acid may be a test sample, and the second nucleic acid may be a portion of the nucleic acid sequence set forth in SEQ ID No. 1. Hybridization of the first and second nucleic acids is conducted under stringent conditions, from low stringency to high stringency, e.g., at a temperature and/or salt content, which tend to disfavor hybridization of noncomplementary nucleotide sequences. Appropriate stringency conditions which promote DNA hybridization, for example, 6. OX sodium chloride/sodium citrate (SSC) at about 45° C, followed by a wash of 2. OX SSC at 50° C are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N Y. (1989), 6.3.1-6.3.6. For example, the salt concentration in the wash step can be selected from a low stringency of about 2. OX SSC at 50° C to a high stringency of about 0.2X SSC at 50° C. In addition, the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22° C to high stringency conditions, at about 65° C. It will be appreciated, however, that although reference herein is made to nucleic acids capable of hybridizing under stringent conditions, hybridization in the practice of the present invention need not actually be conducted under such conditions. The following Specific Example further describes the present invention.

SPECIFIC EXAMPLE 1 Discussion Although the gene encoding the ?-CT subunit has been identified in the plastid genome, until the present invention, CT activity had not been purified, and the σ-CT subunit had not been identified. Progress in this area has been hindered by the tendency of the plastidial ACCase to lose activity upon purification and by the lack of a convenient assay for CT. To further characterize CT, an improved enzymatic assay was therefore developed to be used in the isolation and characterization of the CT subunit. The assay measures CT activity by way of the biotin-dependent decarboxylation of malonyl-CoA to form acetyl-CoA (the reverse reaction of ACCase). Rather than measure the loss of acid stable ¹ C from malonyl- CoA as in previous assays, the procedure described herein directly measures the formation of acetyl-CoA. The reaction is stopped by the addition of hydroxylamine which results in the formation of hydroxamic acid derivatives of both malonyl-CoA and acetyl-CoA. The hydroxamates, acetyl-hydroxa ate (AH) and malonyl- hydroxamate (MH), are easily separated by TLC resulting in unambiguous identification of both substrate and products of the reaction, as seen in Figure 1 B, where "-B" indicates the absence of biotin methyl ester. The formation of the product is completely dependent on the presence of biotin methyl ester as the carboxyl group acceptor, and is linear with time and enzyme. The CT activity of gel- permeation purified ACCase preparations are 1-5 % of ACCase activity. This relatively low activity can most likely be attributed to the use of biotin methyl ester rather than BCCP as acceptor and because the back rather than forward reaction is measured.

A pea cDNA, IEP96, was obtained which has sequence similarity to the σ-CT subunit of E. co//^' ACCase. Hirsh, S., et al., Plant Mol. Biol. 27:1 173-1181 (1995). The first 300 amino acids of the proposed mature IEP96 protein is 47% identical and 68% similar to the 35 kDa σ-CT subunit of E. coli ACCase. Li, S.J. et al. J. Biol. Chem. 267:16841-16847 (1992). However, the remaining amino acid sequence (positions 346-873) of IEP96 is unrelated to other ACCase sequences and instead resembles several cytoskeleton proteins such as integrin, myosin heavy chain and US01 , a cytoskeleton component in yeast involved in intracellular protein trafficking from the ER to the Golgi. Nakajima, H., et al., J. Cell Biol. 113:245-260 (1991). Moreover, because there are numerous carboxylation reactions in plants which are catalyzed by proteins with related sequences, IEP96 may have represented a subunit of any of a number of enzymes.

To further determine the relationship, if any, between IEP96 and ACCase, antibodies were raised to an N-terminal 404 amino acid sequence of the IEP96 protein expressed in E. coli. Western blot analysis of pea chloroplast proteins using IEP96 antibodies detected a protein band with a molecular weight of 91 kd which ran, in SDS-PAGE, at the exact distance as the in vitro translated and chloroplast- imported IEP96 protein (data not shown). As described below, these antibodies were used in combination with biochemical analyses to demonstrate that the IEP96 protein is the σ subunit of pea chloroplast-ACCase.

Results Gel permeation chromatography. Pea chloroplast proteins were fractionated on a Sephacryl S-300 HR gel permeation column and fractions were assayed for ACCase and CT activities (Figure 1A). Both enzyme activities eluted between fractions 27 and 32 with the highest activity in fraction 29. However, in contrast to ACCase activity, CT activity was also detected between fractions 32 and 44 with 30% of maximum activity in fraction 34. Subsequently, CT activity dropped gradually, reaching 2.5% of maximum activity in fraction 44. Figure 1 C sets forth the Western blot analysis of the active fractions (27-32), which revealed the highest levels of BC, BCCP, β-Cl and IEP96 proteins coinciding with highest ACCase activity. Therefore, the co-elution of IEP96 with CT activity and ACCase activity and its subunits (BCCP, BC, and β-Cl) suggested that IEP96 may be a component of the heteromeric-ACCase enzyme. While both the β-Cl and IEP96 protein levels gradually decreased after fraction 32, the BC and BCCP protein levels dropped at a faster rate. These results correlate with the detection of CT activity, but not ACCase activity, in fractions 32 to 44 and may indicate that the ACCase subunits are undergoing partial dissociation during chromatography. The gel permeation elution patterns described above were reproducible throughout at least six independent pea chloroplast isolations.

Immunoprecipitation. To further test if IEP96 is a subunit of pea ACCase and its possible association with the ?-CT subunit, antibodies against IEP96 and β- CT subunit were used to immunoprecipitate gel permeation column fractions having the highest ACCase and CT activities. Figure 2 shows the immunoprecipitation of pea chloroplast ACCase and CT activities by β-Cl and IEP96 (σ-CT) antibodies. Figures 2A and 2B show the percent of maximum ACCase or CT activities remaining after treatment with antibodies to IEP96 (σ-CT), β-Cl or pre-immune sera (Pre σ- CT). Figure 2B shows the resolution of the proteins immunoprecipitated with antibodies to IEP96 (σ-CT), β-Cl, and pre-immune sera (Pre σ-CT) by 7.5% SDS-

PAGE and the probing of these proteins with antibodies to either IEP96 (σ-CT) or β-Cl). One skilled in the art will note that in Figures 2A and 2B, the IEP96 antibodies, but not pre-immune IgG, precipitated both CT and ACCase activities.

When the immunoprecipitates formed with anti-IEP96 were further separated by SDS-PAGE and probed with antibodies to β-Cl, it could be demonstrated that the anti-IEP96 antibodies also co-precipitated β-Cl. In the converse experiment, β-Cl antibodies were found to precipitate both CT and ACCase activities with the concomitant precipitation of the IEP96 protein. Thus, these data further support the hypothesis that IEP96 protein is tightly associated with β-Cl and is a subunit of pea chloroplast ACCase. These results are also consistent with the previous observation of precipitation of an unidentified 91-kd protein using β-Cl antibodies. Sasaki., Y., et al., J. Biol. Chem. 268:25118-255123 (1993).

DEAE chromatography. Figure 3 provides the DEAE analysis of gel permeation purified chloroplast ACCase, with Figure 3A showing CT and ACCase activities in fractions 6 and 9 after mixing, and Figure 3B showing that individual DEAE column fractions were resolved by 10% SDS-PAGE, blotted to nitrocellulose and probed with BC, biotin (BCCP) and IEP96 (σ-CT) antibodies. The association of IEP96 with β-Cl was stable when gel permeation purified ACCase was fractionated by anion-exchange chromatography. The peak of ACCase and CT activity from the gel permeation analysis (fractions 27-31 , Figure 1 ) was applied to a DEAE column and proteins were eluted with a salt concentration gradient. Fractions eluting from the column were assayed for ACCase and CT activities and also resolved by SDS-PAGE and probed using antibodies to BC, biotin, IEP96 and β-Cl. Western blot analysis of column fractions (Figure 3B) indicated that the BC and BCCP proteins have similar elution profiles with the peaks of both proteins eluting in fraction 6. The β-Cl and IEP96 proteins eluted later in the gradient with the peak for both proteins occurring in fraction 9. Although CT activity was detected at low levels in fraction 9 (Figure 3A), almost all ACCase activity was lost upon DEAE chromatography as observed previously. Sasaki., Y., et al., J. Biol. Chem. 268:251 18-255123 (1993) and Alban, C, et al., Biochem. J. 300:557-565 (1994). However, when fractions 6 and 9 were mixed, ACCase activity could be reconstituted and CT activity was enhanced. Therefore, these two fractions must contain all of the subunits needed for the recovery of ACCase and CT activities. Furthermore, the presence of the BC/BCCP containing fractions enhances the CT activity. These results are similar to the recent report that pea BC activity may be higher in the presence of CT subunits. Alban, C., et al. Plant Physiol. 109:927-935 (1995).

Two dimensional PAGE analysis. To further demonstrate that stable complexes are formed between IEP96 and β-Cl, and between BC and BCCP, the gel permeation-purified ACCase (fraction 30, Figure 1 ) was analyzed by two dimensional PAGE analysis (Figure 4). In duplicate gels, proteins were resolved in the first dimension in a 4-10% gradient native polyacrylamide gel and in the second dimension by 8% SDS-PAGE. After blotting to nitrocellulose, one membrane was probed with BC and IEP96 antibodies while the other membrane was probed with β-Cl and biotin (BCCP) antibodies. Both BC and BCCP were detected at the same location in the first dimension, running close to the well. Furthermore, the β- CT and IEP96 proteins migrated together in the first dimension, entering about 2 cm into the gel, between MW markers thyroglobulin (669 kd) and ferritin (440 kd). The mature IEP96 has a calculated pl (from its primary sequence) of 9.7 and therefore, at pH 8.5-8.7 (running buffer) this protein is positively charged and would not be expected to enter the native gel. Interestingly, after in vitro translation and chloroplast import, ³⁵S-labeled IEP96 did not enter the first dimension (data not shown). These results suggest that the endogenous IEP96 protein migrates into the native gradient gel only if it is in a complex with other proteins. Based on this data, another protein with which IEP96 associates is the ϊ-CT subunit of pea ACCase. In addition to the migration of the major spots shown in Figure 4, minor amounts of IEP96 and β-Cl proteins were also found to partially overlap the BC/BCCP complex in the first dimension. This may suggest that a minor association was stably maintained between all four subunits (IEP96, β-Cl, BC, and BCCP) during the native PAGE run.

Pea chloroplast ACCase dissociates into two complexes. These data demonstrate that pea chloroplast-ACCase dissociates into two complexes (β- CT/IEP96 and BC/BCCP) that can be resolved by either DEAE chromatography or native PAGE. These results extend the observation of Alban, C, et al., Biochem J. 300:557-565 (1994), that pea ACCase could be dissociated into two inactive fractions by differential (NH₄)₂S0₄ precipitation and that the activity could be restored by simple recombination of the two fractions. Thus, the accumulated data suggest that, unlike in E. coli, a complex of all four subunits of pea ACCase remains associated during gel filtration and can be at least partially reconstituted from components separated by salt or anion-exchange fractionation. In addition, the BC/BCCP complex and the β-Cllα-Cl complexes are more stable than the entire 600-700-kd ACCase complex. Of these, the β-Cllα-Cl complex is clearly the most stable, and this is perhaps related to the strikingly different pl values. The calculated pl of the pea β-Cl subunit is 4.1 whereas the σ-CT subunit pl is 9.7. Based on their dissociation by salt, anion-exchange chromatography and PAGE, the BC/BCCP complex most likely interacts with the β-Cllα-Cl complex through ionic interactions.

Recently, BC activity was purified from pea chloroplasts. Alban, C, et al., Plant Physiol. 109:927-935 (1995). It was reported that the structure of the active BC corresponded to a complex made up of BCCP with a MW of 38 kd and a BC polypeptide of 31 kd. The 31 -kd polypeptide is substantially different in size from the 50-kd polypeptide previously reported to be the BC subunit of the pea ACCase. Shorrosh, B.S., et al., Plant Physiol. 108:805-812 (1995) and Roesler, K.R., et al., Planta, 198, in press (1995). The data here show that BCCP with MW of 38 kd is in a tight association with BC polypeptide with MW of 50 kd. Therefore, the 31 -kd polypeptide is perhaps a degradation product of the 50 kd polypeptide, another isoform of BC, or an unidentified protein. The carboxyltransferase is membrane associated. It was previously reported that the BC subunit of ACCase is in the soluble fraction of pea chloroplasts. Shorrosh, B.S., et al., Plant Physiol. 108:805-812 (1995). However, Sasaki, Y., et al., J. Biol. Chem. 268:255118-255123 (1993), localized the β-Cl subunit of ACCase to the thylakoid membrane and early reports (Kannangara, C.G., et al., Arch. Biochem. Biophys. 152:83-91 (1972)) reported the association of BCCP with membranes. In an attempt to understand this difference, total pea leaf proteins were extracted and extracts fractionated into soluble and membrane fractions via centrifugation (Figure 5). Figure 5 shows the Western blot analysis of the pea leaf proteins, which were fractionated into supernatant (S) and pellet (P) fractions by centrifugation at 8000 g. After resuspending the pellet in the same volume as the supernatant, equivalent volumes of these fractions (containing 149 μg of supernatant protein and 57 μg of resuspended pellet protein) were resolved on 10% SDS-PAGE, blotted to nitrocellulose, and probed with preimmune sera (Pre), BC, β-Cl, and IEP96 (σ-CT) antibodies. β-Cl (86 kd) and IEP96 (91 kd) were recovered primarily in the insoluble membrane fraction, whereas BC was found in both fractions but at higher levels in the supernatant fraction. Apparently, the BC subunit is more susceptible than the other subunits to solubilization during extraction. Furthermore, by probing inner and outer envelope preparations with antibodies raised to IEP96 the report of Hirsch, S., et al., Plant Mol. Biol. 27:1173-1181 (1995) was confirmed that IEP96 is associated with the inner membrane of pea chloroplast envelopes (data not shown). Thus, the data confirm that pea ACCase is at least partly associated with chloroplast membranes under the extraction conditions described here. These results may also relate to recent observations that osmotically lysed chloroplasts retain an assembly of enzymes capable of light dependent fatty acid synthesis from acetate. Roughan, P.G., et al., Plant Physiol, in press, 110:1239-1247 (1996).

Native structure of pea ACCase. To date, four subunits of the heteromeric ACCase have been characterized and cloned. The deduced mature molecular weights of β-Cl (pea), BC (tobacco), BCCP (Arabidopsis), and σ-CT (IEP96) (pea) are 67, 51 , 21 , and 91 kd, respectively, whereas the apparent molecular weights determined by SDS-PAGE are 86 (pea), 51 (pea), 38 (pea), and 91 kd (pea), respectively. Therefore, if no other protein subunits are involved, the plant heteromeric ACCase would have a native holoenzyme mass of 230 kd (based on the deduced molecular weights) or 266 kd (based on the apparent molecular weights). If plant heteromeric ACCase has the same organization of subunits as found in E. coli (two of each subunit) then the native complex would have a mass in the range of 460-532 kd. This is somewhat lower than the MW of 600-700 kd determined by gel permeation chromatography. Alternatively, if three of each subunit make up the native ACCase then the native MW could be 690 to 798 kd. Therefore, either the chloroplast ACCase elutes anomalously during gel filtration chromatography, has other unidentified subunits, or its molar subunit stoichiometry is different than that of E. coli ACCase.

Other possible functions of lEP96 protein. The deduced IEP96 protein is approximately twice the size of the E. coli σ-CT and a putative red algal σ-CT (Genbank Accession number Z33874). The first 300 amino acids of the proposed mature IEP96 protein share 47% identity and 68 - 71 % similarity with E. coli and red algal σ-CT protein sequences. However, the remaining amino acid sequence (positions 346-873) of IEP96 is unrelated to other ACCase sequences and instead resembles several cytoskeleton proteins such as integrin, myosin heavy chain and USO1 , a cytoskeleton component in yeast involved in intracellular protein trafficking from the ER to the Golgi. Nakajima, H., et al., J. Cell. Biol. 1 13:245-260 (1991 ). Such proteins are characterized by coiled-coil helical structures, and heptapeptide repeats having hydrophobic amino acids in every fourth and seventh position.

Interestingly, a structural motif program (GCG motif) identified in IEP96 a motif (IVIGEGGSGGALAIGC) similar to a prokaryotic membrane lipoprotein lipid attachment site. In prokaryotes, membrane lipoproteins are synthesized with a precursor signal peptide, which is cleaved by a specific lipoprotein signal peptidase (Signal Peptidase II). The peptidase recognizes the consensus sequence (D,E,R,K,){6 residues} (L,A) {2 residues} (I) (G) C and cleaves upstream of the cysteine residue to which a glyceride-fatty acid lipid is attached. Hayashi, S., et al., J. Bioenergetics and Biomembranes 22:451-471 (1990). It would be of interest to determine if IEP96 is modified by a glyceride-fatty acid lipid and whether it is attached to the membrane through such a group, or whether such a modification is involved in the regulation of ACCase. Further work is needed to address these possibilities and the role of the C-terminal region in the pea σ-CT. In conclusion, by both biochemical and immunological criteria it was demonstrated that IEP96 is the σ-carboxyltransferase subunit of the pea chloroplast- ACCase. Thus, all four subunits known to be essential for ACCase activity in prokaryotes have now been identified in higher plant plastids.

Materials and Methods Production of anti-IEP96 serum. The plasmid pisa96 (Hirsch, S., et al.,

Plant Mol. Biol. 27:1173-1181 (1995)) was cut with Nde\IBam \ enzymes to release a cDNA fragment encoding the first 404 amino acids of IEP96 protein. This fragment was subcloned into pET15b at the Λ/ctel/SamHI sites and used to transform BL21 (DE3) cells (Novagen, Madison, Wl). The expression of the 404 amino acids in BL21 cells was induced by treatment with 1 mM isopropyl- ?-D-thiogalacto-pyranoside (dioxane free) for 2 h at 37° C. The expressed protein was resolved by SDS-PAGE and stained with Coomassie Blue. The protein of interest was excised, destained, rinsed with water, and then used to immunize female New Zealand White rabbits (Cocalico Biologicals, Inc.)

Enzymatic assays. ACCase assay is based on the acetyl-CoA dependent formation of acid-stable radioactive malonyl-CoA from H¹ C0₃ ^" and acetyl-CoA. Sauer, A., et al., Naturforsch 39C:268-275 (1984); Alban, C., et al., Biochem. J. 300:557-565 (1994).

To determine carboxyltransferase activity, [2-¹⁴Cjmalonyl-CoA (50 //Ci/μmol) was prepared from [1-¹⁴C]acetate using pea chloroplast extracts as described by Roughan, G., Biochem. J. 300:355-358 (1994). Carboxyl transfer from [2- ¹⁴C]malonyl-CoA to free d-biotin methyl ester was determined in a 20//L reaction containing 5 mM biotin methyl ester, 50 mM Tris pH 8.1 , 12500 d.p.m. of [2- ¹⁴C]malonyl-CoA, and 6 μl enzyme. After 15 min at 30° C, 5 μ of 2 M neutral hydroxylamine was added. After mixing, the reaction mixture was allowed to stand at room temperature for 15 min and then spotted (10 to 20 μL) onto Cellulose 300, F-254 (Selecto Scientific) and analyzed by TLC. The separation of radiolabeled [1- ¹⁴C]acetyl-hydroxamate and [2-¹⁴C]malonyl-hydroxamate was achieved by an 8:2:3 mixture of butanol, acetic acid, and water. [1-¹⁴C]acetyl-hydroxamate produced by this reaction was visualized and counted by digital autoradiography (Instantlmager, Packard Inc.). In some cases, radioactive areas were cut from the TLC plates and quantified in a liquid-scintillation counter. A disadvantage of the assay is that the TLC application and separation of the modified substrate and product require approximately 6 h. Also, the modified product (acetylhydroxamate) evaporates with very long storage at room temperature. Fractionation of pea chloroplast proteins by gel permeation chromatography. Chloroplasts were isolated from pea [Pisum sativum cv little marvel (Burpee)] seedlings as described by Roughan, P.G., Methods Enzymol. 148:327-337 (1987). Chloroplasts were lysed in 50 mM Hepes (pH 8.0), 0.1 % Triton X-100, 10% glycerol, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 1 mM benzamidine hydrochloride, and 5 mM e-amino-n-caproic acid. Complete lysis was achieved by freezing the resuspended chloroplasts at -80° C. The stromal fraction, recovered after centrifugation at 37000 g for 30 min, was loaded onto a HiPrep 16/60 Sephacryl S-300 HR column (Pharmacia, Uppsala, Sweden) and equilibrated with lysis buffer minus Triton X-100 (Solution A). Fractions were collected at 1.5 ml/fraction and assayed for ACCase and CT activities, and used for immunoprecipitations and immunoblots. Protein concentrations were determined by the Bradford, M.M., Anal. Biochem. 72:248-254 (1976) method using bovine gamma globulin as a standard.

Immunoprecipitation analysis. Immunoglobulin G (IgG) was prepared from both immune and preimmune sera using diethylaminoethyl (DEAE)-cellulose chromatography. The IgG immune and preimmune sera (200 μg of each) were lyophilized and resuspended in solution B [solution A (described above) containing 0.25% (w/v) gelatin], 1 % (w/v) casein, and 2% (w/v) BSA] and incubated at 4° C for 1 h to overnight. IgG-sorb with binding capacity of 2 mg/mL IgG (The Enzyme Center, Maiden, MA, USA) was mixed with an equal volume of solution B and incubated at 4° C for 1 h to overnight. Subsequently, the blocked IgG-sorb was aliqated into fractions that can bind at least 60 μg of IgG, and pelleted down by centrifugation. The blocked immune or pre-immune IgG (10, 30, and 60 μg) was incubated with the enzyme (40 μL gel permeation fraction) in a total volume of 64 μL (made up in solution B) for 1 h on ice. Subsequently, the antigen-antibody complexes were mixed with the IgG-sorb pellets and stored on ice for 30 min. The antigen-antibody complexes were precipitated by centrifugation at 14000 g and the supernatant was assayed for ACCase and CT activities. The protein pellets were boiled in SDS sample buffer (Laemmli, U.K. Nature, 227:680-685 (1970)), resolved by SDS-PAGE, and blotted to nitrocellulose. Two dimensional gel electrophoresis and blotting procedures. After resolving chloroplast proteins by gel permeation chromatography, 50 μL from fraction 30 were loaded onto a 4-10% gradient native polyacrylamide gel using a vertical mini-gel system with an 8 X 7-cm cell format. Proteins were resolved at 82 V for 6 h at 4° C. Subsequently, the lane of interest was excised and equilibrated in SDS sample buffer for 15 min. The second dimension was in 8% SDS-PAGE using vertical large gel system with a 14 X 11-cm format. Analysis in the second dimension was performed essentially as described by O'Farrell, P.H., J. Biol. Chem. 10:4007-4021 (1975) except that the native gel slice was placed in direct contact with the stacking gel of the SDS system without the use of agarose. Also, the native gel was flanked at both ends with wells, which included prestained molecular weight markers (Gibco BRL). _ <■ -. _

Ion-exchange chromatography. Chloroplast proteins resolved by gel permeation chromatography (1 mL each of fractions 27 to 31 ) were pooled and loaded on a 1 mL RESOURCE Q column (Pharmacia) equilibrated with solution C (20 mM Tris pH 8.5 / 10% glycerol). Protein fractions (20 fractions) were collected at 1.0 mL/fraction using a buffer gradient formed by mixing solution C with the elution buffer D (solution C plus 0.5 M NaCI). At the end of the gradient, solution C containing 1 M NaCI was used to elute any remaining proteins bound to the column. An aliquot (38 μL each) from collected fractions was resolved by SDS-PAGE and immunoblotted. Protein extraction for immunoblot analysis Pea leaves (1 g) were homogenized with a Polytron on ice in 1 mL of 100 mM Tris pH 7.4 containing 1 mM PMSF and 1 mM benzamidine hydrochloride. After centrifugation at 8000 g the supernatant was mixed and boiled in SDS sample buffer (total volume of 1.32 mL) and the pellet was boiled in 1.32 mL of SDS sample buffer. A volume of supernatant containing 149 μg protein and an equal volume of resuspended pellet containing 57 μg protein were loaded onto a 10% SDS-PAGE and blotted to nitrocellulose.

Immunoblot analysis. Proteins were resolved by 8 or 10% SDS-PAGE (Laemmli, U.K. Nature, 227:680-685 (1970)), transferred to nitrocellulose using a Pharmacia Novablot system and reversibly stained with Ponceau S. The nitrocellulose membrane was blocked in Tris-buffered saline [10 mM Tris, 0.9% (w/v) NaCI, 2.5% (w/v) BSA] and probed with either pre-immune or immune serum diluted in Tris-buffered saline containing 0.05% (v/v) Tween 20 and 1 % (w/v) BSA. BC (Shorrosh, B.S., et al., Plant Physiol. 108:805-812 (1995)), /?-CT (Sasaki., Y., et al., J. Biol. Chem. 268:25118-255123 (1993)), biotin (Sigma, St. Louis, MO) and 1EP96 antibodies were used at 1 :500, 1 :1000, 1 :1000, and 1 :500 dilutions, respectively. Subsequently, antigen-antibody complexes were visualized using alkaline phosphatase-conjugated anti-(rabbit IgG).

The foregoing discussion discloses and describes merely exemplary embodiments of the present invention. One skilled in the art will readily recognize from such discussion, and from the accompanying drawings, that various changes, modifications and variations can be made therein without departing from the spirit and scope of the invention.

All publications referred to herein are expressly incorporated by reference. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Ohlrogge, John B. Shorrosh, Basil S.

(ii) TITLE OF INVENTION: Structure and Expression of the alpha-Carboxyltransferase Subunit of Heteromeric-Acetyl -CoA Carboxylase

(in) NUMBER OF SEQUENCES: 2

(ιv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE. Harness, Dickey & Pierce PLC

(B) STREET: 5445 Corporate Drive

(C) CITY- Troy

(D) STATE: MI

(E) COUNTRY USA

(F) ZIP. 48098

(v) COMPUTER READABLE FORM

(A) MEDIUM TYPE Floppy disk

(B) COMPUTER- IBM PC compatible

(C) OPERATING SYSTEM- PC-DOS/MS-DOS

(D) SOFTWARE Patentin Release #1 0, Version #1 25

(vi) CURRENT APPLICATION DATA-

(A) APPLICATION NUMBER-

(B) FILING DATE.

(C) CLASSIFICATION:

(vni) ATTORNEY/AGENT INFORMATION (A) NAME. Smith, DeAnn F. (C) REFERENCE/DOCKET NUMBER 655000006PCA

(ix) TELECOMMUNICATION INFORMATION

(A) TELEPHONE: 810-641-1600

(B) TELEFAX: 810-641-0270

(C) TELEX- 287637

(2) INFORMATION FOR SEQ ID NO : 1 :

(i) SEQUENCE CHARACTERISTICS.

(A) LENGTH: 3274 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY linear

(ii) MOLECULE TYPE. cDNA

(m) HYPOTHETICAL: NO

(iv) ANTI-SENSE. NO

(vi) ORIGINAL SOURCE

(A) ORGANISM: Pisum sativum (I) ORGANELLE Chloroplast ( i x) FEATURE :

(A) NAME/KEY: CDS

(B) LOCATION: 259..2886

(D) OTHER INFORMATION, /function- "Malonyl CoA syntesis from Acetyl CoA" /product= "a-CT Subunit of Pea Chloroplast ACCase"

(x) PUBLICATION INFORMATION-

(A) AUTHORS: Shorrosh, Basil S.

Savage, Linda J. Soil , Jurgen Ohlrogge, John B.

(B) TITLE: The Pea Chloroplast Membrane-Associated

Protein, IEP96. Is a Subumt of Acetyl-CoA Carboxylase

(C) JOURNAL- Plant Journal (G) DATE- 1996

(K) RELEVANT RESIDUES IN SEQ ID NO : 1 FROM 1 TO 3274

(xi) SEQUENCE DESCRIPTION- SEQ ID NO-1-

TTCAACCCCA ATCTCATTCA GTCATTCCTC TCACTCCTAC TACCAACAAT AACAACTCCC 60

TCπTCππ CCCCTTCATT GACGATCTCC ACGAAGCTAC AATACCCTTA TAATπCAAC 120

AACTCTTTCT CTTTCTATTA CCCATTTTCT TCTπCTCTC TCTACCTCTC TTTCCTCCTT 180

GGTTTTGGAT CTTCAAπTC TCATTCACTG ATπTGGTGA TTTGAGGAAT TGAGATTGAA 240

AGACTGTGπ GAGGAACT ATG GCT TCC TCT TCT GCA ACT CTT GTT GGT TCT 291

Met Ala Ser Ser Ser Ala Thr Leu Val Gly Ser 1 5 10

ACT GCT TCT GAT CTT CTC AGG AGT TCA ACT ACT GGT TTC ACT GGT GTC 339 Thr Ala Ser Asp Leu Leu Arg Ser Ser Thr Thr Gly Phe Thr Gly Val 15 20 25

CCT πG AGA ACC πG GGA AGG GCA GGG πG GTC TTG AAA AGA AGG GAT 387 Pro Leu Arg Thr Leu Gly Arg Ala Gly Leu Val Leu Lys Arg Arg Asp 30 35 40 πA ACT GTT AGT GTT ACT GCT AAG TTG AGG AAG GTG AAG AGG CGT GAA 435 Leu Thr Val Ser Val Thr Ala Lys Leu Arg Lys Val Lys Arg Arg Glu 45 50 55

TAT CCA TGG TCA AGT AAC CCT GAT CCC AAT ATG AAA GGT GGG CGG πG 483 Tyr Pro Trp Ser Ser Asn Pro Asp Pro Asn Met Lys Gly Gly Arg Leu 60 65 70 75

CGT CAT CTC TCA ACG TTC CAG CCA CTC AAA CAG CCG CCA AAG CCT Gπ 531 Arg His Leu Ser Thr Phe Gin Pro Leu Lys Gin Pro Pro Lys Pro Val 80 85 90

ATT TTG GAG Tπ GAA AAG CCT CTT Aπ AAT ATG GAA AAG AAG ATT AAT 579 He Leu Glu Phe Glu Lys Pro Leu He Asn Met Glu Lys Lys He Asn 95 100 105

GAT TTT CGG AAG GTG GCA GAA AAA ACT GGT GTG GAT TTA AGT GAT CAG 627 Asp Phe Arg Lys Val Ala Glu Lys Thr Gly Val Asp Leu Ser Asp Gin 110 115 120 Aπ CTC GCA TTG GAG GCT AAG TAC CAA AAG GCT TTG GTG GAA TTG TAT 675 He Leu Ala Leu Glu Ala Lys Tyr Gin Lys Ala Leu Val Glu Leu Tyr 125 130 135

ACA AAT CTA ACT CCT ATA CAG CGG GTC ACC GTT GCA CGG CAT CCT AAC 723 Thr Asn Leu Thr Pro He Gin Arg Val Thr Val Ala Arg His Pro Asn 140 145 150 155

AGG CCT ACT πC CTG GAT CAC ATG TAT AAC ATG ACT GAA AAG Tπ GTG 771 Arg Pro Thr Phe Leu Asp His Met Tyr Asn Met Thr Glu Lys Phe Val 160 165 170

GAA CTC CAT GGT GAT CGT GAA GGA TAC GAT GAT CCT GCT ATT GCC GCT 819 Glu Leu His Gly Asp Arg Glu Gly Tyr Asp Asp Pro Ala He Ala Ala 175 180 185

GGT CTA GGG AGT ATA GAT GGT AAA ACC TAC ATG TTC ATC GGC CAC CAA 867 Gly Leu Gly Ser He Asp Gly Lys Thr Tyr Met Phe He Gly His Gin 190 195 200

AAG GGT AGA GAT ACT AAA GAA AAT ATT AAG CGT AAC TTT GCG ATG CCA 915 Lys Gly Arg Asp Thr Lys Glu Asn He Lys Arg Asn Phe Ala Met Pro 205 210 215

ACT CCA CAC GGT TAT AGG AAA GCT CTG CGC TTG ATG GAA TAT GCA GAT 963 Thr Pro His Gly Tyr Arg Lys Ala Leu Arg Leu Met Glu Tyr Ala Asp 220 225 230 235

CAT CAC GGG TTC CCG ATA GTT ACT TTC ATT GAC ACC CCT GGG GCA TTT 1011 His His Gly Phe Pro He Val Thr Phe He Asp Thr Pro Gly Ala Phe 240 245 250

GCT GAC CTC AAA TCC GAG CAA CTT GGT CAA GGT GAA GCA ATT GCT CAT 1059 Ala Asp Leu Lys Ser Glu Gin Leu Gly Gin Gly Glu Ala He Ala His 255 260 265

AAT πG AGA TCC ATG TTT GCT CTG AAG GTG CCA GTT ATT TCT ATA GTT 1107 Asn Leu Arg Ser Met Phe Ala Leu Lys Val Pro Val He Ser He Val 270 275 280

ATT GGC GAA GGT GGA TCT GGC GGT GCC CTT GCC Aπ GGA TGT GCT AAT 1155 He Gly Glu Gly Gly Ser Gly Gly Ala Leu Ala He Gly Cys Ala Asn 285 290 295

AAA πA CTC ATG Cπ GAA AAT TCA GTG TTC Tπ Gπ GCC ATG CCA GAG 1203 Lys Leu Leu Met Leu Glu Asn Ser Val Phe Phe Val Ala Met Pro Glu 300 305 310 315

GCA TGC GGT GCA ATC πG TGG AAG AGT AAT AAA GCT GCT CCA AAG GCT 1251 Ala Cys Gly Ala He Leu Trp Lys Ser Asn Lys Ala Ala Pro Lys Ala 320 325 330

GCT GAG CGA CTG AAG Aπ ACA GCA TCT GCA TTG πG GAT TTG GAA ATT 1299 Ala Glu Arg Leu Lys He Thr Ala Ser Ala Leu Leu Asp Leu Glu He 335 340 345

GCA GAT GGC Aπ ATA CCG GAG CCC CTG GCT GGT GCA CAT ACT GAT CCA 1347 Ala Asp Gly He He Pro Glu Pro Leu Ala Gly Ala His Thr Asp Pro 350 355 360 AGT TGG ATG TCT CAA CAG ATT AAA Aπ GCA ATC AAT GAA GCT ATG GAT 1395 Ser Trp Met Ser Gin Gin He Lys He Ala He Asn Glu Ala Met Asp 365 370 375

GAA CTC ACC AAG πG AGC ACA GAA GAC CTA ATA AAA GAT CGC ATG CAT 1443 Glu Leu Thr Lys Leu Ser Thr Glu Asp Leu He Lys Asp Arg Met His 380 385 390 395

AAG TTC CGA AAA CTC GGT GTT GAT GGG ATC CAG GAA GGA Aπ CCT πA 1491 Lys Phe Arg Lys Leu Gly Val Asp Gly He Gin Glu Gly He Pro Leu 400 405 410

GTT CCC AGT AAG AAA GTC AAC ACG AAA AAG AGG GAA ATA GGT GTT CCG 1539 Val Pro Ser Lys Lys Val Asn Thr Lys Lys Arg Glu He Gly Val Pro 415 420 425

CCG AAG AGG CAG GAG GTA CCT Aπ CCT GAT TCT CAA ATA GAG GCT GAA 1587 Pro Lys Arg Gin Glu Val Pro He Pro Asp Ser Gin He Glu Ala Glu 430 435 440

ATT GAG AAA CTG AAG AAA GCT ATT TTC GAA GGG GAG GAC TCT TCT GCG 1635 He Glu Lys Leu Lys Lys Ala He Phe Glu Gly Glu Asp Ser Ser Ala 445 450 455

GCA AAG AAG AAT CCT GGT TCT CAA ATA GGG TCT GCA ATT GAC AAA CTG 1683 Ala Lys Lys Asn Pro Gly Ser Gin He Gly Ser Ala He Asp Lys Leu 460 465 470 475

AAG GGT TTA Tπ TTG GAA GGT AAG GAC TCT TCT GCG GCA AAG AAG ACT 1731 Lys Gly Leu Phe Leu Glu Gly Lys Asp Ser Ser Ala Ala Lys Lys Thr 480 485 490

CCT GGT TCT CAA ATA GTG GCT GAA CTT GAC AAA CTG AAG GGT TTA TAT 1779 Pro Gly Ser Gin He Val Ala Glu Leu Asp Lys Leu Lys Gly Leu Tyr 495 500 505

TTG GAA GCT AAG GAC TCT TCT GCG GCA AAG GTT CCT GGT TCT CAA ATA 1827 Leu Glu Ala Lys Asp Ser Ser Ala Ala Lys Val Pro Gly Ser Gin He 510 515 520

GTG GCT GAA ATT GAG AAA CTG AAG AAT AGT ATT TTC GAA GAT GAG GAC 1875 Val Ala Glu He Glu Lys Leu Lys Asn Ser He Phe Glu Asp Glu Asp 525 530 535

TCC TCT TCT GCT GTT CTG CCA GAG AAG ATT CCT GGT TCT GAA ATA GCG 1923 Ser Ser Ser Ala Val Leu Pro Glu Lys He Pro Gly Ser Glu He Ala 540 545 550 555

Gπ GAA ATT GCG AAA CTG AAG AAA AAT ATT TTG GAA GGT AAG GAC TCC 1971 Val Glu He Ala Lys Leu Lys Lys Asn He Leu Glu Gly Lys Asp Ser 560 565 570

TCT TCT GAG CCT TCA AAA CTC GAT CTG GAC AAG ACA ATA GAG ACT CTG 2019 Ser Ser Glu Pro Ser Lys Leu Asp Leu Asp Lys Thr He Glu Thr Leu 575 580 585

AAA AGG GAG GTT AAT CGA GAA TTC TCT GAG GCC GTT AAA GCC GCG GGC 2067 Lys Arg Glu Val Asn Arg Glu Phe Ser Glu Ala Val Lys Ala Ala Gly 590 595 600 TTA ACA AAA ACA πG ACG AAA CTA CGG GGT GAA ATT TCA AAA GCA AAG 2115 Leu Thr Lys Thr Leu Thr Lys Leu Arg Gly Glu He Ser Lys Ala Lys 605 610 615

GCA GGT AAC CAA CCT TTG ACT CCA TTG CTG AAG GTG GAG ATA AAA AGT 2163 Ala Gly Asn Gin Pro Leu Thr Pro Leu Leu Lys Val Glu He Lys Ser 620 625 630 635

Tπ AAC CAA AGG TTA TCA GCG GCT CCT AAT TCC AGA AAG CTG CTG AAG 2211 Phe Asn Gin Arg Leu Ser Ala Ala Pro Asn Ser Arg Lys Leu Leu Lys 640 645 650

AAG CGT GGC TTG TTA AGA GAA GTG ACT AAA GTC AAG CTT TTG TTG GAT 2259 Lys Arg Gly Leu Leu Arg Glu Val Thr Lys Val Lys Leu Leu Leu Asp 655 660 665

AAA AAC AAG GCT GCA ACA CGT AAG CAA GAG CTA AAG AAA AAG TCG GAT 2307 Lys Asn Lys Ala Ala Thr Arg Lys Gin Glu Leu Lys Lys Lys Ser Asp 670 675 680

GAA CAC AAG GAG GCT GCA AGA CTT GAG CAA GAA CTA AAG AAA AAG TTT 2355 Glu His Lys Glu Ala Ala Arg Leu Glu Gin Glu Leu lys Lys Lys Phe 685 690 695

GAT GAA GTC ATG GAT ACT CCT AGA ATA AAG GAA AAG TAT GAA GCA TTA 2403 Asp Glu Val Met Asp Thr Pro Arg He Lys Glu Lys Tyr Glu Ala Leu 700 705 710 715

CGG TCT GAA GTC CGG CGC GTT GAC GCA TCC TCA GGA AGT GGC TTG GAC 2451 Arg Ser Glu Val Arg Arg Val Asp Ala Ser Ser Gly Ser Gly Leu Asp 720 725 730

GAT GAA CTG AAG AAG AAA ATC ATT GAG πC AAT AAG GAG GTA GAC TTG 2499 Asp Glu Leu Lys Lys Lys He He Glu Phe Asn Lys Glu Val Asp Leu 735 740 745

GAG CTG GCT ACA GCT GTG AAG TCG GTA GGG TTA GAG GTT GAG TCT GTG 2547 Glu Leu Ala Thr Ala Val Lys Ser Val Gly Leu Glu Val Glu Ser Val 750 755 760

AAA CCA GGA CAT GGC TGG AAC AAG TCT TCA GTG CCA GAG ATA GAA GAA 2595 Lys Pro Gly His Gly Trp Asn Lys Ser Ser Val Pro Glu He Glu Glu 765 770 775

CTA AAC AAA GAT GTA CAA AAG GAA Aπ GAA ATT GTG GCA AAC TCG TCA 2643 Leu Asn Lys Asp Val Gin Lys Glu He Glu He Val Ala Asn Ser Ser 780 785 790 795

CCG AAT GTT AAG AGA CTG ATA GAG CAA TTG AAA CTG GAG GTT GCC AAG 2691 Pro Asn Val Lys Arg Leu He Glu Gin Leu Lys Leu Glu Val Ala Lys 800 805 810

TCT GGA GGG AAA CCA GAT TCT GAA TCG AAG AGT AGA ATT GAT GCT TTG 2739 Ser Gly Gly Lys Pro Asp Ser Glu Ser Lys Ser Arg He Asp Ala Leu 815 820 825

ACG CAA CAG Aπ AAG AAG AGC CTT GCT GAG GCT GTT GAT TCG CCT AGC 2787 Thr Gin Gin He Lys Lys Ser Leu Ala Glu Ala Val Asp Ser Pro Ser 830 835 840 CTG AAA GAG AAG TAT GAA AAC CTC ACT CGA CCA GCA GGA GAC ACT CTC 2835 Leu Lys Glu Lys Tyr Glu Asn Leu Thr Arg Pro Ala Gly Asp Thr Leu 845 850 855

ACC GAT GAC AAA πG AGA GAG AAA GTT GGT GTA AAT CGC AAC TTC TCT 2883 Thr Asp Asp Lys Leu Arg Glu Lys Val Gly Val Asn Arg Asn Phe Ser 860 865 870 875

TAATAGACTG CTGCTCGTGC GGAGCTGGTG AAGACGGAGC TGAGAGTGGA AACTGCATCA 2943

GAGTCTTCCT GATGATATAT TπACCAATA TCπCATGGT CAATAAπGT TAGAAAGAGA 3003

GTTAAAGGAG ATACAGGACG AπTGTGGCT GATATTπTC ATCATCTAGA CTACAAGCGC 3063

TAATAAAACA TAAAACCATT TTGTATCTGT TGAGTTAGAA TGTTGCAGTT AATTπAGAA 3123

TGACTTGCπ TATCTACGTC TTTTTTCTTC CCTππCAA AAATTTTAAG TGTTTTCCCT 3183

GAATAGACAT AGATATAAAT TAπTGTGπ TGTAGAACAT AATAAATAAC ATGATGCTGT 3243

GGTGπTGAA TGTCTTATπ GCAAGAAAAA A 3274

(2) INFORMATION FOR SEQ ID NO 2

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH 875 amino acids

(B) TYPE amino acid (D) TOPOLOGY linear

(n) MOLECULE TYPE protein

(xi) SEQUENCE DESCRIPTION SEQ ID NO 2

Met Ala Ser Ser Ser Ala Thr Leu Val Gly Ser Thr Ala Ser Asp Leu 1 5 10 15

Leu Arg Ser Ser Thr Thr Gly Phe Thr Gly Val Pro Leu Arg Thr Leu 20 25 30

Gly Arg Ala Gly Leu Val Leu Lys Arg Arg Asp Leu Thr Val Ser Val 35 40 45

Thr Ala Lys Leu Arg Lys Val Lys Arg Arg Glu Tyr Pro Trp Ser Ser 50 55 60

Asn Pro Asp Pro Asn Met Lys Gly Gly Arg Leu Arg His Leu Ser Thr 65 70 75 80

Phe Gin Pro Leu Lys Gin Pro Pro Lys Pro Val He Leu Glu Phe Glu 85 90 95

Lys Pro Leu He Asn Met Glu Lys Lys He Asn Asp Phe Arg Lys Val 100 105 110

Ala Glu Lys Thr Gly Val Asp Leu Ser Asp Gin He Leu Ala Leu Glu 115 120 125

Ala Lys Tyr Gin Lys Ala Leu Val Glu Leu Tyr Thr Asn Leu Thr Pro 130 135 140 Ile Gin Arg Val Thr Val Ala Arg His Pro Asn Arg Pro Thr Phe Leu 145 150 155 160

Asp His Met Tyr Asn Met Thr Glu Lys Phe Val Glu Leu His Gly Asp 165 170 175

Arg Glu Gly Tyr Asp Asp Pro Ala He Ala Ala Gly Leu Gly Ser He 180 185 190

Asp Gly Lys Thr Tyr Met Phe He Gly His Gin Lys Gly Arg Asp Thr 195 200 205

Lys Glu Asn He Lys Arg Asn Phe Ala Met Pro Thr Pro His Gly Tyr 210 215 220

Arg Lys Ala Leu Arg Leu Met Glu Tyr Ala Asp His His Gly Phe Pro 225 230 235 240

He Val Thr Phe He Asp Thr Pro Gly Ala Phe Ala Asp Leu Lys Ser 245 250 255

Glu Gin Leu Gly Gin Gly Glu Ala He Ala His Asn Leu Arg Ser Met 260 265 270

Phe Ala Leu Lys Val Pro Val He Ser He Val He Gly Glu Gly Gly 275 280 285

Ser Gly Gly Ala Leu Ala He Gly Cys Ala Asn Lys Leu Leu Met Leu 290 295 300

Glu Asn Ser Val Phe Phe Val Ala Met Pro Glu Ala Cys Gly Ala He 305 310 315 320

Leu Trp Lys Ser Asn Lys Ala Ala Pro Lys Ala Ala Glu Arg Leu Lys 325 330 335

He Thr Ala Ser Ala Leu Leu Asp Leu Glu He Ala Asp Gly He He 340 345 350

Pro Glu Pro Leu Ala Gly Ala His Thr Asp Pro Ser Trp Met Ser Gin 355 360 365

Gin He Lys He Ala He Asn Glu Ala Met Asp Glu Leu Thr Lys Leu 370 375 380

Ser Thr Glu Asp Leu He Lys Asp Arg Met His Lys Phe Arg Lys Leu 385 390 395 400

Gly Val Asp Gly He Gin Glu Gly He Pro Leu Val Pro Ser Lys Lys 405 410 415

Val Asn Thr Lys Lys Arg Glu He Gly Val Pro Pro Lys Arg Gin Glu 420 425 430

Val Pro He Pro Asp Ser Gin He Glu Ala Glu He Glu Lys Leu Lys 435 440 445

Lys Ala He Phe Glu Gly Glu Asp Ser Ser Ala Ala Lys Lys Asn Pro 450 455 460 Gly Ser Gin He Gly Ser Ala He Asp Lys Leu Lys Gly Leu Phe Leu 465 470 475 480

Glu Gly Lys Asp Ser Ser Ala Ala Lys Lys Thr Pro Gly Ser Gin He 485 490 495

Val Ala Glu Leu Asp Lys Leu Lys Gly Leu Tyr Leu Glu Ala Lys Asp 500 505 510

Ser Ser Ala Ala Lys Val Pro Gly Ser Gin He Val Ala Glu He Glu 515 520 525

Lys Leu Lys Asn Ser He Phe Glu Asp Glu Asp Ser Ser Ser Ala Val 530 535 540

Leu Pro Glu Lys He Pro Gly Ser Glu He Ala Val Glu He Ala Lys 545 550 555 560

Leu Lys Lys Asn He Leu Glu Gly Lys Asp Ser Ser Ser Glu Pro Ser 565 570 575

Lys Leu Asp Leu Asp Lys Thr He Glu Thr Leu Lys Arg Glu Val Asn 580 585 590

Arg Glu Phe Ser Glu Ala Val Lys Ala Ala Gly Leu Thr Lys Thr Leu 595 600 605

Thr Lys Leu Arg Gly Glu He Ser Lys Ala Lys Ala Gly Asn Gin Pro 610 615 620

Leu Thr Pro Leu Leu Lys Val Glu He Lys Ser Phe Asn Gin Arg Leu 625 630 635 640

Ser Ala Ala Pro Asn Ser Arg Lys Leu Leu Lys Lys Arg Gly Leu Leu 645 650 655

Arg Glu Val Thr Lys Val Lys Leu Leu Leu Asp Lys Asn Lys Ala Ala 660 665 670

Thr Arg Lys Gin Glu Leu Lys Lys Lys Ser Asp Glu His Lys Glu Ala 675 680 685

Ala Arg Leu Glu Gin Glu Leu Lys Lys Lys Phe Asp Glu Val Met Asp 690 695 700

Thr Pro Arg He Lys Glu Lys Tyr Glu Ala Leu Arg Ser Glu Val Arg 705 710 715 720

Arg Val Asp Ala Ser Ser Gly Ser Gly Leu Asp Asp Glu Leu Lys Lys 725 730 735

Lys He He Glu Phe Asn Lys Glu Val Asp Leu Glu Leu Ala Thr Ala 740 745 750

Val Lys Ser Val Gly Leu Glu Val Glu Ser Val Lys Pro Gly His Gly 755 760 765

Trp Asn Lys Ser Ser Val Pro Glu He Glu Glu Leu Asn Lys Asp Val 770 775 780 Gin Lys Glu He Glu He Val Ala Asn Ser Ser Pro Asn Val Lys Arg 785 790 795 800

Leu He Glu Gin Leu Lys Leu Glu Val Ala Lys Ser Gly Gly Lys Pro 805 810 815

Asp Ser Glu Ser Lys Ser Arg He Asp Ala Leu Thr Gin Gin He Lys 820 825 830

Lys Ser Leu Ala Glu Ala Val Asp Ser Pro Ser Leu Lys Glu Lys Tyr 835 840 845

Glu Asn Leu Thr Arg Pro Ala Gly Asp Thr Leu Thr Asp Asp Lys Leu 850 855 860

Arg Glu Lys Val Gly Val Asn Arg Asn Phe Ser 865 870 875

Claims

WE CLAIM:

1. A vector comprising an isolated nucleic acid molecule encoding an a- carboxyltransferase subunit of plant acetyl-CoA carboxylase.

2. A plant cell transformed with the vector of Claim 1.

3. The vector of Claim 1 , wherein the nucleic acid molecule comprises a nucleotide sequence capable of hybridizing under stringent conditions with the complement of SEQ ID No. 1.

4. The vector of Claim 1 , wherein the isolated nucleic acid molecule comprises a nucleotide sequence that encodes the polypeptide of SEQ ID No. 2.

5. A plant transfected with the vector of Claim 1.

6. Seed of the plant of Claim 5 comprising the nucleic acid molecule encoding an σ-carboxyltransferase subunit of acetyl-CoA carboxylase.

7. A method for increasing carboxylation of acetyl-CoA in a plant comprising the steps of: a) introducing a DNA construct comprising an isolated nucleic acid molecule encoding an a- carboxyltransferase subunit of plant acetyl-CoA carboxylase into a plant cell; and b) growing the cell into a plant.

8. The method of Claim 7, wherein the nucleic acid molecule comprises a nucleotide sequence capable of hybridizing under stringent conditions with the complement of SEQ ID No. 1.

9. The method of Claim 7, wherein the nucleic acid molecule comprises a nucleotide sequence that encodes the polypeptide of SEQ ID No. 2.

10. A transgenic plant produced by the method of Claim 7.

11. A transgenic plant produced by breeding the plant of Claim 10, wherein the plant retains the trait of increased carboxylation of acetyl-CoA.

12. The seeds of the plant of Claim 10.

13. The seeds of the plant of Claim 11.

14. A method of producing seeds of an oilseed plant variety wherein the seeds have increased oil content comprising the steps of: a) introducing a DNA construct comprising an isolated nucleic acid molecule encoding an σ- carboxyltransferase subunit of plant acetyl-CoA carboxylase into a plant cell; b) growing the cell into a plant; and c) harvesting the seeds of the plant of step b).

15. The method of Claim 14, wherein the nucleic acid molecule comprises a nucleotide sequence capable of hybridizing under stringent conditions with the complement of SEQ ID No. 1.

16. The method of Claim 14, wherein the nucleic acid molecule comprises a nucleotide sequence that encodes the polypeptide of SEQ ID No. 2.

17. The seeds of Claim 14.

18. A method for producing herbicide resistant Gramineae plants comprising the step of introducing an isolated nucleic acid molecule encoding an σ-carboxyltransferase subunit of plant acetyl-CoA carboxylase into a plant cell and growing the cell into a plant.

19. The method of Claim 18, wherein the nucleic acid molecule comprises a nucleotide sequence capable of hybridizing under stringent conditions with the complement of SEQ ID No. 1.

20. The method of Claim 18, wherein the nucleic acid molecule comprises a nucleotide sequence that encodes the polypeptide of SEQ ID No. 2.

21. A transgenic plant produced by the method of Claim 18.

22. A transgenic plant produced by breeding the plant of Claim 21 , wherein the plant retains the trait of herbicide resistance.

23. The seeds of the plant of Claim 21.

24. The seeds of the plant of Claim 22.