WO2000001828A1

WO2000001828A1 - A modified arabidopsis thaliana cac1, cac2 or cac3 promoter and an arabidopsis thaliana cac1, cac2 or cac3 suppressor element and methods of use thereof

Info

Publication number: WO2000001828A1
Application number: PCT/US1999/014968
Authority: WO
Inventors: Basil J. Nikolau; Eve S. Wurtele; Joong-Kook Choi; Abbaraju Hari Kishan Rao
Original assignee: Iowa State University Research Foundation, Inc.
Priority date: 1998-07-01
Filing date: 1999-07-01
Publication date: 2000-01-13
Also published as: AU4965399A

Abstract

The present invention provides a modified Arabidopsis thaliana CAC1, CAC2 or CAC3 promoter, the Arabidopsis thaliana CAC1, CAC2 or CAC3 suppressor element, a recombinant vector comprising such a modified promoter or such a suppressor element, a host cell comprising such a recombinant vector, methods of using the modified promoter or the suppressor element to regulate or suppress, respectively, expression of a coding sequence in a cell, a plant cell in which the expression of a coding sequence in the plant cell has been so regulated or suppressed, as well as a plant tissue, a plant organ and a plant comprising such a plant cell. Also provided by the present invention is an isolated or purified nucleic acid molecule comprising the genomic DNA sequence of the carboxyltransferase α subunit (CAC3) of the acetyl CoA carboxylase (ACC), a vector comprising such a sequence, a cell transformed with such a vector, a plant cell, a plant tissue, a plant organ or a plant in which the level of a subunit of ACC, ACC enzymatic activity or malonyl CoA has been altered, and a method of producing such a plant cell, plant tissue, plant organ or plant.

Description

A MODIFIED ARABIDOPSIS THALIANA CAC1, CAC2 or CAC3 PROMOTER

AND AN ARABIDOPSIS THALIANA CAC1, CAC2 or CAC3 SUPPRESSOR

ELEMENT AND METHODS OF USE THEREOF

STATEMENT OF GOVERNMENT SUPPORT The present invention was made, in part, with funding from the United States Department of Agriculture under agency contract no. USDA 9701912. Therefore, the United States of America may have certain rights in the invention.

TECHNICAL FIELD OF THE INVENTION The present invention relates to a modified promoter, a suppressor element, a recombinant vector comprising a modified promoter or a suppressor element, a host cell comprising such a recombinant vector, methods of using the modified promoter or the suppressor element to regulate or suppress, respectively, expression of a coding sequence in a cell, a plant cell in which the expression of a coding sequence in the plant cell has been so regulated or suppressed, as well as a plant tissue, plant organ and plant comprising such a plant cell. The present invention also relates to an isolated or purified nucleic acid molecule comprising the genomic DNA sequence of the carboxyltransferase α subunit (coded by the CAC3 gene) of the acetyl CoA carboxylase (ACC), a vector comprising such a sequence, and a cell transformed with such a vector. The present invention further relates to a plant cell, a plant tissue, a plant organ or a plant in which the level of a subunit of ACC, ACC enzymatic activity or malonyl CoA has been altered, and a method of producing such a plant cell, plant tissue, plant organ or plant.

BACKGROUND OF THE INVENTION The first step in the biosynthesis of fatty acids in bacteria, yeast, plants

(including chloroplasts) and animals is a primary regulatory or rate-limiting reaction catalyzed by the enzyme ACC. ACC catalyzes the ATP-dependent carboxylation of acetyl CoA to malonyl CoA. The reaction is actually a two-step process, which requires a biotin prosthetic group and which involves the carboxylation of an enzyme- bound biotin molecule to form a carboxybiotin-enzyme complex and the transfer of the carboxyl group to acetyl CoA. In plants, malonyl CoA not only is a common intermediate in the biosynthesis of fatty acids but is a common intermediate in the pathway leading to the biosynthesis of a variety of phytochemicals, including cuticular waxes, flavonoids, stilbenoids, anthroquinones, malonated D-amino acids, N-malonyl-ACC and malonic acid. Most of the specialized phytochemicals are synthesized in the cytosol, whereas de novo fatty acid biosynthesis occurs in the plastids.

There are two forms of ACC in plants. One form is monomeric, nondissociable, cytosolic (although a homomeric ACC of similar structure is present in the plastids of some plants, such as maize and wheat) and comprises a single type of biotin-containing polypeptide that ranges in size from 220 to 260 kd. The monomeric polypeptide comprises biotin carboxylase, biotin carboxyl carrier protein, and carboxyltransferase domains. The other form is heteromeric, dissociable, plastidic, and comprises the subunits biotin carboxylase (coded by the CAC2 gene), biotin carboxyl carrier (coded by the CAC1 gene), carboxyltransferase α (coded by the CAC3 gene), and carboxyltransferase β (coded by the accD gene). The CAC1 (chloroplastic acetyl coA carboxylase), CAC2 and CAC3 genes are in the nuclear genome of the plant cell, whereas the accD gene is in the plastidic genome. It has now been surprisingly and unexpectedly discovered that the CAC1, CAC2 and CAC3 genes, as isolated from Arabidopsis thaliana, comprise promoters that comprise suppressor elements. It also has been surprisingly and unexpectedly discovered that the promoters, upon modification of the suppressor elements contained therein, constitute strong promoters. In view of this discovery, it is an object of the present invention to provide a modified Arabidopsis thaliana CAC1, CAC2 or CAC3 promoter. It is another object of the present invention to provide an Arabidopsis thaliana CAC1, CAC2 or CAC3 suppressor element. Providing a recombinant vector comprising the modified promoter or the suppressor element and a host cell comprising such a recombinant vector are other objects of the present invention. It is yet another object of the present invention to provide methods of using the modified promoter or the suppressor element to regulate or suppress, respectively, expression of a coding sequence in the nucleus of a cell. In this regard, it is still yet another object of the present invention to provide a plant cell in which the expression of a coding sequence in the nucleus of the plant cell has been so regulated or suppressed, as well as a plant tissue, a plant organ and a plant comprising such a plant cell. Other objects of the present invention include the provision of an isolated or purified nucleic acid molecule comprising the CAC3 genomic DNA sequence, as well as a vector comprising such a sequence, and a cell transformed with such a vector. A further object of the present invention is to provide a method of altering the level of a subunit of ACC, ACC enzymatic activity, or malonyl CoA in a plant cell, plant tissue, plant organ or plant as well as the plant cell, plant tissue, plant organ or plant so produced. These and other objects and advantages of the present invention will become apparent to one of ordinary skill in the art from the following description.

BRIEF SUMMARY OF THE INVENTION

The present invention provides an isolated or purified nucleic acid fragment consisting essentially of a modified promoter of the CAC1, CAC2 or CAC3 gene of Arabidopsis thaliana. The promoter comprises bases from about -1048 to about -1 relative to the translation start codon of the CAC1 gene and is modified in the region from about -785 to about -529 relative to the translation start codon so as to inactivate a suppressor element present in that region, comprises bases from about -1412 to about -1 relative to the translation start codon of the CAC2 gene and is modified in the region from about -880 to about -586 relative to the translation start codon so as to inactivate a suppressor element present in that region, or comprises bases from about -1561 to about -1 relative to the translation start codon of the CAC3 gene and is modified in the region from about -422 to about -1,000 relative to the translation start codon so as to inactivate a suppressor element present in that region. Preferably, the promoter is modified in the indicated region by (i) a deletion, (ii) an insertion, or (iii) a substitution of sufficient size and appropriate position to inactivate the suppressing effect of the suppressor element present in that region.

The present invention also provides an isolated or purified nucleic acid fragment consisting essentially of a suppressor element of the promoter of the CAC1, CAC2 or CAC3 gene of Arabidopsis thaliana. The suppressor element comprises bases from about -785 to about -529 relative to the translation start codon of the CAC1 gene, from about -880 to about -586 relative to the translation start codon of the CAC2 gene, or from about -422 to about -1,000 relative to the translation start codon of the CAC3 gene.

Further provided by the present invention is a recombinant vector comprising the modified promoter or the suppressor element operably linked to a coding sequence to be expressed in a cell. In this regard, a host cell comprising such a recombinant vector is also provided. Still further provided by the present invention are methods of using the modified promoter or the suppressor element to regulate or suppress, respectively, the expression of a coding sequence in a cell. In one embodiment, the method comprises contacting the cell with a vector comprising an isolated or purified nucleic acid fragment consisting essentially of a modified promoter of the CAC1, CAC2 or CAC3 gene of Arabidopsis thaliana (as described above) operably linked to the coding sequence, wherein the nucleic acid fragment regulates the expression of the coding sequence in the cell. In another embodiment, the method comprises randomly inserting an isolated or purified nucleic acid fragment consisting essentially of a modified promoter of the CAC1, CAC2 or CAC3 gene of Arabidopsis thaliana (as described above) into the genome of the cell, whereupon a given random insertion, the expression of a coding sequence in the cell is regulated by the nucleic acid fragment. In yet another embodiment, the method comprises suppressing the expression of a coding sequence in the cell by randomly inserting an isolated or purified nucleic acid fragment consisting essentially of a suppressor element (as described above) into the genome of the cell, whereupon a given random insertion, the expression of a coding sequence in the cell is suppressed. In still yet another embodiment, the method comprises suppressing the expression of a coding sequence in the cell by nonrandomly inserting an isolated or purified nucleic acid fragment consisting essentially of a suppressor element (as described above) into the genome of the cell, whereupon targeted insertion, the expression of the coding sequence in the cell is suppressed. Accordingly, the present invention further provides a plant cell in which the expression of a coding sequence has been regulated or suppressed in accordance with one of the above methods, as well as a plant tissue, plant organ or plant comprising such a plant cell. The present invention also provides an isolated or purified nucleic acid molecule comprising the genomic DNA sequence of a carboxyltransferase α subunit of a plant ACC, such as that which is isolated from Arabidopsis, and a continuous fragment thereof comprising at least about 20 nucleotides, wherein the continuous fragment is not derived entirely from an exon of the genomic DNA sequence. Preferably, the isolated or purified nucleic acid molecule comprises SEQ ID NO: 3, a continuous fragment thereof comprising at least about 20 nucleotides, wherein the continuous fragment is not derived entirely from an exon of the genomic DNA sequence or a nucleic acid molecule comprising a genomic DNA sequence of a carboxyltransferase α subunit of a plant ACC that hybridizes to either one of the foregoing under stringent conditions. Also provided is an isolated or purified nucleic acid molecule encoding a modified carboxyltransferase α subunit of a plant ACC and a continuous fragment thereof comprising at least about 20 nucleotides. Accordingly, the present invention also provides a vector comprising an isolated or purified nucleic acid molecule as described above and a host cell comprising such a vector. Accordingly, in another embodiment of the present invention, a method of altering the level of a subunit of ACC in a plant cell, a plant tissue, a plant organ or a plant is provided. The method comprises contacting the plant cell, plant tissue, plant organ or plant with a vector comprising a gene encoding a subunit of ACC selected from the group consisting of biotin carboxyl carrier, biotin carboxylase, carboxyltransferase α and carboxyltransferase β, wherein the gene comprises a modified promoter as described herein. The vector increases or decreases the level of the subunit in the plant cell, plant tissue, plant organ or plant. Preferably, the alteration of the level of the subunit results in an alteration of the level of ACC enzymatic activity in the plant cell, plant tissue, plant organ or plant. More preferably, the alteration of the level of the subunit results in an alteration of the level of malonyl CoA in the plant cell, plant tissue, plant organ or plant. Accordingly, the present invention further provides a plant cell, a plant tissue, a plant organ and a plant in which the level of the subunit of ACC, ACC enzymatic activity or malonyl CoA has been altered in accordance with the method.

BRIEF DESCRIPTION OF THE FIGURES Fig. 1 is the nucleic acid sequence of the CAC1 promoter of Arabidopsis thaliana [SEQ ID NO: 1].

Fig. 2 is the nucleic acid sequence of the CAC2 promoter of Arabidopsis thaliana [SEQ ID NO: 2].

Figs. 3 A and 3B combined are the genomic DNA sequence of the CAC3 gene of Arabidopsis thaliana [SEQ ID NO: 3], wherein exons are indicated by capital letters and the translation start codon is underlined.

DETAILED DESCRIPTION OF THE INVENTION The present invention is predicated on the surprising and unexpected discovery that the promoters of the Arabidopsis thaliana CAC1, CAC2 and CAC3 genes comprise a suppressor element. The present invention is further predicated on the surprising and unexpected discovery that the CAC1 promoter, upon modification of the suppressor element contained therein, constitutes a strong promoter.

The cloning and sequencing of the Arabidopsis thaliana CAC1 cDNA and gene is described in Choi et al., Plant Physiol. 109: 619-625 (1995), and Ke et al., Plant Physiol. 113: 1091-1100 (1997). The mRNA is 1.1 kb.

The cloning and sequencing of the Arabidopsis thaliana CAC2 cDNA and gene is described in Sun et al., Plant Physiol. 115: 1371-1383 (1997). The mRNA is 2.0 kb. The cloning and sequence of the Arabidopsis thaliana CAC3 cDNA is described in Shorrosh et al., /α«t J. 10: 261-268 (1996). The mRNA is 2.85 kb. The cloning and sequencing of the Arabidopsis thaliana CAC3 genomic DNA is described herein.

The cloning and sequencing oϊaccD from pea is described in Sasaki et al., J. Biol. Chem. 268: 25118-25123 (1993). The GenBank Accession No. is AF056971. In view of the above, the present invention provides an isolated or purified nucleic acid fragment consisting essentially of a modified promoter of the CAC1, CAC2 or CAC3 gene of Arabidopsis thaliana. By "isolated" is meant the removal of a nucleic acid from its natural environment. By "purified" is meant that a given nucleic acid, whether one that has been removed from nature (including genomic DNA and mRNA) or synthesized (including cDNA) and/or amplified under laboratory conditions, has been increased in purity, wherein "purity" is a relative term, not "absolute purity."

An isolated or purified nucleic acid fragment consisting essentially of the CACl, CAC2 or CAC3 promoter can be obtained in accordance with the methods set forth in Example 3, for example. The promoter comprises bases from about -1048 to about -1 relative to the translation start codon of the CACl gene, from about -1412 to about -1 relative to the translation start codon of the CAC2 gene, or from about -1561 to about -1 relative to the translation start codon of the CAC3 gene. Alternatively, the nucleic acid fragment consisting essentially of the CACl, CAC2 or CAC3 promoter can be synthesized in accordance with methods known in the art. The promoter is "modified" in that the suppressing effect of a suppressor element present in the region from about -785 to about -529 relative to the translation start codon of the CACl gene, from about -880 to about -586 relative to the translation start codon of the CAC2 gene, or from about -422 to about -1,000 relative to the translation start codon of the CAC3 gene is inactivated. Preferably, the promoter is modified in the indicated region by (i) a deletion, (ii) an insertion or (iii) a substitution of sufficient size and appropriate position to inactivate the suppressing effect of a suppressor element present in the region. One of ordinary skill in the art knows how to generate insertions, deletions and/or substitutions in a given nucleic acid molecule and appreciates that such insertions, deletions and/or substitutions must not be positioned or of such a size as to negate the effect sought to be achieved. Preferably, the deletion and the substitution are at least about one base in size. Preferably, the insertion is at least about 6 bases in size. Optionally, the insertion or substitution can comprise one or more modified and/or nonnaturally occurring base(s). Alternatively, a nucleic acid fragment comprising a modified CACl, CAC2 or CAC3 promoter can be synthesized in accordance with methods known in the art.

The present invention also provides an isolated or purified nucleic acid fragment consisting essentially of a suppressor element of the promoter of the CACl, CAC2 or CAC3 gene of Arabidopsis thaliana, wherein the suppressor element comprises bases from about -785 to about -529 relative to the translation start codon of the CACl gene, from about -880 to about -586 relative to the translation start codon of the CAC2 gene, or from about -422 to about -1,000 relative to the translation start codon of the CAC3 gene. Gel retardation and competition assays indicate that the suppressor element is in the region from about -655 to about -529 relative to the translation start codon of the CACl gene. The suppressor element can be isolated from the CACl, CAC2 or CAC3 promoter, for example, by restriction enzyme digestion of the promoter, such as by digestion of the CACl promoter with Msp I and Hpa I, or can be synthesized in accordance with methods known in the art. In another embodiment, the present invention also provides a vector comprising a nucleic acid fragment as described above operably linked to a coding sequence. By "operably linked" is meant that the modified promoter can promote transcription of the coding sequence or that the suppressor element can suppress transcription of the coding sequence. A nucleic acid fragment as described above can be cloned into any suitable vector and can be used to transform or transfect any suitable host cell. The selection of vectors and host cells and the methods to construct vectors are as described below.

In view of the above, the present invention provides a host cell comprising a recombinant vector as described above. Suitable host cells include those set forth below.

In view of the above, the present invention provides a method of regulating the expression of a coding sequence in a cell, in particular in the nuclear genome of the cell. The method comprises contacting the cell with a vector comprising an isolated or purified nucleic acid fragment consisting essentially of a modified promoter as described above operably linked to the coding sequence. The modified promoter regulates the expression of the coding sequence in the cell. Desirably, the modified promoter is a stronger promoter than that which is operably linked to the coding sequence as it is found naturally. The present invention also provides another method of regulating the expression of a coding sequence in a cell, wherein the method comprises randomly inserting an isolated or purified nucleic acid fragment consisting essentially of a modified promoter as described above into the genome, in particular the nuclear genome of a cell. Upon a given random insertion, the expression of a coding sequence in the cell is regulated by the nucleic acid fragment.

Further provided by the present invention is a method of suppressing the expression of a coding sequence in a cell. The method comprises randomly inserting an isolated or purified nucleic acid fragment consisting essentially of a suppressor element as described above into the genome, in particular the nuclear genome, of the cell. Upon a given random insertion, the expression of a coding sequence in the cell is suppressed by the nucleic acid fragment. Still further provided by the present invention is another method of suppressing the expression of a coding sequence in a cell. The method comprises nonrandomly inserting an isolated or purified nucleic acid fragment consisting essentially of a suppressor element as described above into the genome, in particular the nuclear genome, of the cell. Upon a given targeted insertion, the expression of the targeted coding sequence in the cell is suppressed by the nucleic acid fragment. Vectors as described above can be introduced into a plant by any suitable means as described herein. For example, cells in tissue culture can be transformed with a vector. This method is particularly useful for plants, like maize for example. Arabidopsis, on the other hand, preferably is transformed using the Agrobacterium- mediated infiltration method (see, e.g., Chang et al., Plant J. 5(4): 551-558 (1994); Katavic et al., Molec. Gen. Genet. 245(3): 363-370 (1994); and http ://www.bio.net: 80/hypermail/ARABIDOPSIS/9707/0015.html)).

Methods of random insertion, targeted insertion and random activation of gene expression are known to those of ordinary skill in the art. With respect to random insertion in a plant cell, desirably a nucleic acid fragment consisting essentially of a modified promoter or a suppressor element as described is placed near the ends of the T-DNA of an Agrobacterium Ti-based plant transformation plasmid, which is then used to transform a plant cell in accordance with methods known in the art. With respect to targeted insertion, desirably homologous recombination based gene replacement technology (see, Schnable et al., Curr. Opinions Plant Biol. 1 : 123

(1998); Puchta and Hohn, Trends Plant Sci. 1 : 340 (1996); Kempin et al., Nature 389: 802 (1997)) is used. In view of the above, the present invention provides a plant cell in which the expression of a coding sequence in the plant cell has been regulated in accordance with one of the above methods of regulating the expression of a coding sequence in the plant cell. In addition, the present invention provides a plant tissue, a plant organ or a plant comprising such a plant cell.

Further in view of the above, the present invention also provides a plant cell in which the expression of a coding sequence in the plant cell has been suppressed in accordance with one of the above methods of suppressing the expression of a coding sequence in the plant cell. Accordingly, the present invention also provides a plant tissue, a plant organ or a plant comprising such a plant cell.

The present invention also provides an isolated or purified nucleic acid molecule comprising the genomic DNA sequence of a carboxyl transferase α subunit of a plant ACC, such as that which is isolated from Arabidopsis, and a continuous fragment thereof comprising at least about 20 nucleotides, wherein the continuous fragment is not derived entirely from an exon of the genomic DNA sequence.

Preferably, the isolated or purified nucleic acid molecule comprises SEQ ID NO: 3, a continuous fragment thereof comprising at least about 20 nucleotides, wherein the continuous fragment is not derived entirely from an exon of the genomic DNA sequence, or a nucleic acid molecule comprising a genomic DNA sequence of a carboxyltransferase α subunit of a plant ACC that hybridizes to either one of the foregoing under stringent conditions.

Also provided is an isolated or purified nucleic acid molecule encoding a modified carboxyltransferase α subunit of a plant ACC, which comprises one or more insertions, deletions and/or substitutions, and a continuous fragment thereof comprising at least about 20 nucleotides. Desirably, the modified subunit does not differ functionally from the corresponding unmodified subunit, such as that comprising SEQ ID NO: 3. Preferably, an ACC comprising a modified carboxyltransferase α subunit converts acetyl CoA to malonyl CoA at least about 50%, more preferably at least about 75%, most preferably at least about 90% as well as the corresponding unmodified ACC, as determined by in vitro assay using labeled acetyl CoA, wherein "labeled" means any means of detection, such as a radioactive isotope. With respect to the above, one of ordinary skill in the art knows how to generate insertions, deletions and/or substitutions in a given nucleic acid molecule. Also with respect to the above, "does not differ functionally from" is intended to mean that the modified carboxyltransferase subunit of a plant ACC has enzymatic activity characteristic of the unmodified subunit. In other words, it acts upon the same substrate and generates the same product. The modified enzyme, however, can be more or less active than the unmodified enzyme as desired in accordance with the present invention.

Nucleic acid molecules encoding subunits of ACC can be isolated from any plant source. Suitable plant sources include, but are not limited to, Arabidopsis, soybean, alfalfa, corn, wheat, sorghum, barley, rice, oats, rye, soybean, rapeseed, canola, cotton, safflower, peanut, palm, sorghum, sunflower, beet, and various vegetable and fruit crops, such as cucumber, tomato, peppers, and the like. With respect to the above isolated or purified nucleic acid molecule comprising the genomic DNA sequence of a carboxyltransferase subunit of a plant ACC, it is preferred that the one or more substitutions(s) do(es) not result in a change in an amino acid of the enzyme. Alternatively, and also preferred, is that the one or more substitution(s) result(s) in the substitution of an amino acid with another amino acid of approximately equivalent size, shape and charge. Also with respect to the above isolated or purified nucleic acid molecule, a

"continuous fragment of at least about 20 nucleotides of the isolated or purified nucleic acid molecule, wherein the continuous fragment is not derived entirely from an exon of the CAC3 gene," is a continuous fragment that, for example, comprises intronic and/or regulatory sequences, alone or in further combination with exonic sequences.

The above isolated or purified nucleic acid molecules also can be characterized in terms of "percentage of sequence identity." In this regard, a given nucleic acid molecule as described above can be compared to a nucleic acid molecule encoding a corresponding gene (i.e., the reference sequence) by optimally aligning the nucleic acid sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence, which does not comprise additions or deletions, for optimal alignment of the two sequences. The percentage of sequence identity is calculated by determining the number of positions at which the identical nucleic acid base occurs in both sequences, i.e., the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity. Optimal alignment of sequences for comparison may be conducted by computerized implementations of known algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI, or BlastN and BlastX available from the National Center for Biotechnology Information, Bethesda, MD), or by inspection. Sequences are typically compared using BESTFIT or BlastN with default parameters.

"Substantial sequence identity" means that at least 75%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% of the sequence of a given nucleic acid molecule is identical to a given reference sequence. Typically, two polypeptides are considered to be substantially identical if at least 40%, preferably at least 60%, more preferably at least 90%, and most preferably at least 95% of the amino acids of which the polypeptides are comprised are identical to or represent conservative substitutions of the amino acids of a given reference sequence. Another indication that polynucleotide sequences are substantially identical is if two molecules selectively hybridize to each other under stringent conditions. The phrase "selectively hybridizing to" refers to the selective binding of a single-stranded nucleic acid probe to a single-stranded target DNA or RNA sequence of complementary sequence when the target sequence is present in a preparation of heterogeneous DNA and/or RNA. Stringent conditions are sequence dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 20°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.

In view of the above, "stringent conditions" preferably allow for from about 25% to about 5% mismatch, more preferably from about 15% to about 5% mismatch, and most preferably from about 10% to about 5% mismatch. "At least moderately stringent conditions" preferably allow for from about 40% to about 15% mismatch, more preferably from about 30% to about 15% mismatch, and most preferably from about 20% to about 15% mismatch. "Low stringency conditions" preferably allow for from about 60% to about 35% mismatch, more preferably from about 50% to about 35% mismatch, and most preferably from about 40% to about 35% mismatch. With respect to the preceding ranges of mismatch, 1% mismatch corresponds to one degree decrease in the melting temperature.

One of ordinary skill in the art will appreciate, however, that two polynucleotide sequences can be substantially different at the nucleic acid level, yet encode substantially similar, if not identical, amino acid sequences, due to the degeneracy of the genetic code. The present invention is intended to encompass such polynucleotide sequences.

The above-described nucleic acid molecules can be used, in whole or in part (i.e., as fragments), to identify and isolate corresponding genes from other plants as well as nonplants (e.g., yeast and bacterium) for use in the context of the present inventive method using conventional means as known in the art. For example, such molecules or fragments thereof can be used in chromosome walking, genomic subtraction, which requires the availability of strains having deletions of the target gene (Strauss and Ausubel, PNAS USA 87: 1889-1893 (1990); and Sun et al., Plant Cell 4: 119-128 (1992)), transposon (Chuck et al., Plant Cell 5: 371-378 (1993); Dean et al., Plant J. 2: 69-81 (1992); Grevelding et al., PNAS USA 899: 6085-6089 (1992); Swinburne et al, Plant Cell 4: 583-595 (1992); Fedoroff and Smith, Plant J. 3: 273- 289 (1993); and Tsay et al., Science 260: 342-344 (1993)) and T-DNA tagging (Feldmann, Plant J. 1: 71-82 (1991); Feldmann et al., Science 243: 1351-1354 (1989); Herman et al., Plant Cell 11: 1051-1055 (1989); Konz et al., EMBO J. 9: 1337-1346 (1989); and Kieber et al., Cell 72: 427-441 (1993)), and heterologous probe selection techniques in accordance with methods well-known in the art. Although T-DNA tagging, chromosome walking or heterologous probe selection can identify a DNA fragment that putatively contains the gene of interest, the DNA fragment must be confirmed by genetic complementation or some other means. In another embodiment, the present invention also provides a vector comprising a nucleic acid molecule comprising the genomic DNA sequence of a carboxyltransferase α subunit of a plant ACC or a fragment thereof as described above. A nucleic acid molecule as described above can be cloned into any suitable vector and can be used to transform or transfect any suitable host.

The selection of vectors and methods to construct vectors are commonly known to persons of ordinary skill in the art and are described in general technical references (see, in general, "Recombinant DNA Part D," Methods in Enzymology, Vol. 153, Wu and Grossman, eds., Academic Press (1987)). Desirably, the vector comprises regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host (e.g., bacterium, fungus, plant or animal) into which the vector is to be introduced, as appropriate and taking into consideration whether the vector is DNA or RNA. Preferably, the vector comprises regulatory sequences that are specific to the genus of the host. Most preferably, the vector comprises regulatory sequences that are specific to the species of the host.

Constructs of vectors, which are circular or linear, can be prepared to contain an entire nucleic acid sequence as described above or a portion thereof ligated to a replication system functional in a prokaryotic or eukaryotic host cell. Replication systems can be derived from ColEl, 2 mμ plasmid, λ, SV40, bovine papilloma virus, and the like.

In addition to the replication system and the inserted nucleic acid, the construct can include one or more marker genes, which allow for selection of transformed or transfected hosts. Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation in an auxotrophic host to provide prototrophy, and the like.

Suitable vectors include those designed for propagation and expansion or for expression or both. A preferred cloning vector is selected from the group consisting of the pUC series the pBluescript series (Stratagene, LaJolla, CA), the pET series (Novagen, Madison, WI), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clonetech, Palo Alto, CA). Bacteriophage vectors, such as λ GT10, λGTl 1, λZapII (Stratagene), λ EMBL4, and λ NM1149, also can be used. Examples of plant expression vectors include pBHOl, pBI101.2, pBI101.3, pBI121 and pBLN19 (Clonetech, Palo Alto, CA). Examples of animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clonetech, Palo Alto, CA).

The expression vector optionally further comprises a transit peptide sequence between the promoter and coding sequence. For those genes that are normally expressed in the mitochondrion or plastid, it is preferred that the expression vector comprise a mitochondrial or plastidic transit peptide, respectively. Numerous plant gene products are known to contain transit peptide sequences. For example, the small subunit of ribulose bisphosphate carboxylase, ferredoxin, chlorophyll a/b binding protein, and the like, comprise transit peptide sequences. Such transit peptide sequences can be isolated/synthesized and used in expression vectors in accordance with the present invention. Regardless of the source of the DNA fragment coding for the transit peptide, it should include a translation initiation codon and an amino acid sequence recognized by and functional in the organelle of the host plant cell or plant. In view of the above, the present invention provides a host cell comprising a vector as described above. Suitable hosts include E. coli, B. subtilis, P. aerugenosa, S. cerevisiae, and N crassa. E. coli, in particular E. coli TB-1, TG-2, DH5α, XL- Blue MRF' (Stratagene), SA2821 and Y1090 are preferred hosts. A more preferred host is XL-Blue MRF' or TG02. In yet another embodiment, the present invention provides a method of altering the level of a subunit of acetyl CoA carboxylase in a plant cell, a plant tissue, a plant organ or a plant. The method comprises contacting the plant cell, plant tissue, plant organ or plant with a vector comprising a gene encoding a subunit of ACC selected from the group consisting of biotin carboxyl carrier, biotin carboxylase, carboxyl transferase α and carboxyl transferase β, wherein the gene comprises a modified promoter as described above. The vector increases or decreases the level of a subunit of ACC in the plant cell, plant tissue, plant organ or plant. Preferably, the alteration of the level of the subunit results in an alteration of the level of ACC enzymatic activity in the plant cell, plant tissue, plant organ or plant. More preferably, the alteration of the level of a subunit of ACC results in an alteration of the level of malonyl CoA in the plant cell, plant tissue, plant organ or plant. Accordingly, the present invention further provides a plant cell, a plant tissue, a plant organ and a plant in which the level of a subunit of ACC, ACC enzymatic activity or malonyl CoA has been altered in accordance with the method.

Preferably, the nucleic acid molecule used in the present inventive method is one of those described above. In this regard, nucleic acid molecules that correspond to the above-described plant nucleic acid molecules but which have been isolated from animal, bacterial or yeast sources can be used in the context of the present inventive method to increase the level of a monomeric acetyl CoA carboxylase or a subunit of a heteromeric ACC, ACC enzymatic activity or malonyl CoA in a plant cell, a plant tissue, a plant organ or a plant, provided that a cDNA sequence is used in those instances where the animal, bacterial or yeast genomic sequence contains introns that may not be properly processed in a plant. In addition, it may be necessary to alter the cDNA sequence so that it contains codon sequences that are preferred in plant species over animal, bacterial or yeast species.

Preferred vectors for use in the present inventive method are characterized as described above. Such vectors can be introduced into a plant by any suitable means. For example, cells in tissue culture can be transformed with a vector. This method is particularly useful for plants like maize, for example. Arabidopsis, on the other hand, preferably is transformed using the Agrobacterium-mediated infiltration method (see, e.g., Chang et al., Plant J. 5(4): 551-558 (1994); Katavic et al., Molec. Gen. Genet. 245(3): 363-370 (1994); and http://www.bio.net:80/hypermai ARABIDOPSIS/ 9707/0015.html)).

If it is desired to increase the expression of a monomeric ACC, a subunit of a heteromeric ACC, ACC enzymatic activity or malonyl CoA, it is preferred to do so by introducing a gene encoding a monomeric ACC or a subunit of a heteromeric ACC. The gene is preferably introduced by way of a vector. It is preferred that either multiple extra copies of the gene are introduced into the plant cell, plant tissue, plant organ or plant or that a vector comprising a strong promoter, such as a modified promoter described herein, is introduced into the plant cell, plant tissue, plant organ or plant such that the gene is expressed at a higher rate, thereby generating more mRNA, which, in turn, is translated into more of the encoded enzyme.

Techniques for contacting a plant cell, a plant tissue, a plant organ or a plant with a vector so that the vector is taken up by a plant cell, alone or as part of a plant tissue, a plant organ or a plant, and expressed therein are known in the art. Such methods involve plant tissue culture techniques, for example. Herein, "contacting" is intended to mean that the cell, tissue, organ or plant is brought into contact with the vector in such a manner that the vector enters the cell and is expressed therein. The plant cell, plant tissue, plant organ or plant can be contacted with the vector by any suitable means, including direct transformation, e.g., polyethylene glycol precipitation (Paszkowski et al., EMBO J. 3: 2717-2722 (1984), cell bombardment, i.e., attaching the DNA to metallic pellets and blasting them through the plant's cell wall (Fromm et al., Bio/Technology 8: 833-839 (1990); Gordon-Kamm et al., Plant Cell 2: 603-618 (1990); and Klein et al., Nature 327: 70-73 (1987)).

Exogenous DNA can be introduced into a dicotyledonous plant cell by insertion of the nucleic acid encoding a gene involved in acetyl CoA production into the Ti plasmid of Agrobacterium and adding suitable ingredients to promote transformation thereby (Horsch et al., Science 223: 496-498 (1984); Fraley et al., PNAS USA 80: 4803 (1983); and DeBlock et al., EMBO J. 3: 1681-1689 (1984)). Other techniques are available for the introduction of exogenous DNA into a plant and/or a subset of its constituent cells, including electroporation (Fromm et al., PNAS USA 82: 5824 (1995), microinjection, protoplast-mediated gene transfer, and silicon carbide crystal- mediated gene transfer. These various techniques are discussed in Genetic Engineering News 14(4): at pages 1, 3 and 24, and are generally known in the art. See, for example, Weising et al., Ann. Rev. Genet. 22: 421-477 (1988)).

Transformed plant cells, which are derived by any of the above transformation techniques can be cultured to regenerate a whole plant, which possesses the desired transformed phenotype. Plant regeneration from cultured protoplasts is described in Evans et al., Protoplast Isolation and Culture, Handbook of Plant Cell Culture, MacMillan Publishing Co., New York, pp. 124-176 (1983); and Binding, Regeneration of Plants, Plant Protoplasts, CRC Press, Boca Raton, pp. 21-73 (1985). Regeneration also can be obtained from plant callus, explants, organs or parts thereof. Such regeneration techniques are described generally in Klee et al., Ann. Rev. of Plant Phys. 38: 467-486 (1987).

One of ordinary skill will appreciate that, after an expression cassette is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.

A method of decreasing the level of a monomeric ACC or a subunit of a heteromeric ACC, ACC enzymatic activity or malonyl CoA levels is co-supression. See, for example, Que et al., E>ev. Genet. 22(1): 100-109 (1998) and Smyth, Curr. Biol. 7(12): R793-R795 (1997).

In addition to the above, gene replacement technology can be used to increase or decrease expression of a given gene. Gene replacement technology is based upon homologous recombination (see, Schnable et al., Curr. Opinions Plant Biol. 1: 123 (1998)). The nucleic acid of the enzyme of interest can be manipulated by mutagenesis (e.g., insertions, such as with a suppressor element as described herein, deletions, duplications, or replacements, such as replacement of a promoter with a modified promoter as described herein) to either increase or decrease enzymatic function. The altered sequence can be introduced into the genome to replace the existing, e.g., wild-type, gene via homologous recombination (Puchta and Hohn, Trends Plant Sci. 1: 340 (1996); Kempin et al., Nature 389: 802 (1997)).

Also in addition to the above, organelle re-targeting can be used to increase or decrease expression of a monomeric ACC, a subunit of a heteromeric ACC, ACC enzymatic activity or malonyl CoA production. For example, one of the above- mentioned genes can be modified by removing its organelle-targeting sequence and replacing it with a novel organelle-targeting sequence (see, for example, Roesler et al., Plant Physiol. 113(1): 75-81 (1997) in re retargeting of a cystolic enzyme to the plastids; Moloney et al., Biotechnol. Genet. Eng. Rev. 14: 321-336 (1997); deCastro Silva et al., Plant Mol. Biol 30(4): 769-780 (1996); and Cline et al., Ann. Rev. Cell Dev. Biol. 12: 1-26 (1996)). The altered sequence can then be introduced into the plant genome via standard transformation procedures.

The activity of ACC can be measured by using labeled substrates in vitro. See for example, Nikolau et al., Arch Biochem. Biophys. 211 : 605-612 (1981), and Nikolau et al, Arch. Biochem. Biophys. 228: 86-96 (1984). Malonyl CoA concentrations can be determined using methods described in Roughan, Biochem. J. 327: 267-273 (1997), and Anderson et al., Plant Physiol. 118: 1127-1138 (1998). In view of the above method, the present invention also provides a bacterium, a yeast, an animal, including a cell, tissue or organ thereof, or a plant, including a cell, tissue or organ thereof, in which the level of a monomeric ACC, a subunit of a heteromeric ACC, ACC enzymatic activity or malonyl CoA has been altered in accordance with the above-described method. Preferably, the present inventive method is used to generate a plant cell, a plant tissue, a plant organ or a plant. The plant cell can be cultured and kept as plant tissue culture cells or certain plant hormones known in the art can be added to the culture medium, thereby causing the plant tissue culture cells to differentiate and thereby form a new plant variety. Such plant culturing methods useful in the performance of this aspect of the invention are well known in the art. Accordingly, the present invention also provides a plant cell, a plant tissue, a plant organ and a plant in which the level of a monomeric ACC, a subunit of a heteromeric ACC, ACC enzymatic activity or malonyl CoA has been altered. In addition to being useful in the study of malonyl CoA generation in plants, including the spatial and temporal patterns of expression of ACC, the above-described methods are useful in the generation of plants for the production of malonyl CoA- derived phytochemicals. The methods are useful in the alteration of the level of a monomeric acetyl CoA carboxylase as well as a subunit of a heteromeric ACC, ACC enzymatic activity or malonyl CoA levels in plants, including wild-type and mutant plants, such as alfalfa, corn, wheat, sorghum, barley, rice, oats, rye, soybean, rapeseed, canola, cotton, safflower, peanut, palm, sunflower, beet, and various vegetable and fruit crops, such as cucumber, tomato, peppers, and the like. By "alteration" is meant that a monomeric ACC, a subunit of a heteromeric ACC, ACC enzymatic activity or malonyl CoA level in a given plant (or plant cell, tissue or organ) is different as a result of the practice of a present inventive method as compared to a like plant, the monomeric ACC, a subunit of a heteromeric ACC, ACC enzymatic activity or malonyl CoA level of which has not been altered as a result of the practice of a present inventive method. However, the above-described methods, as well as the nucleic acid fragments, recombinant vectors and host cells, are useful in the study of the expression of any coding sequence from any host cell and are useful in the regulation/suppression of the expression of a coding sequence in other contexts, such as disease states.

The above method can be adapted for in vitro production of malonyl CoA, which, in turn, can be used to produce malonyl CoA phytochemicals. For example, the various enzymes required for malonyl CoA synthesis can be prepared from a suitable host and placed in a reaction vessel with suitable substrates, an energy source, co-factors and other ingredients known in the art so as to produce malonyl CoA.

EXAMPLES The present invention is described further in the context of the following examples. These examples serve to illustrate further the present invention and are not intended to limit the scope of the invention.

Example 1 This example describes the accumulation of the CACl, CAC2, CAC3 and accD niRNAs in Arabidopsis siliques at between 1 and 15 days after flowering (i.e., from pre-fertilization to the onset of dessication) using Northern blot hybridization.

RNA was extracted from developing Arabidopsis siliques as described previously (Weaver et al., Plant Physiol. 110: 1021 (1995)). The RNA concentrations were determined from the absorbance at 280 and 260 nm. ³²P-labeled CACl, CAC2, CAC3 and accD RNAs were obtained by in vitro transcription of pBSK clones. Ten μ g of RNA from each tissue sample were fractionated by electrophoresis in formaldehyde-containing agarose gels. After transfer of the RNA to nylon membranes, hybridizations were conducted in a buffer containing 50% formamide at 65°C for 12-16 hr using ³²P-labeled RNA probes. Hybridized membranes were rinsed twice with 2 x SSC, 2% SDS for 10 min at room temperature, and then washed twice with 0.1 x SSC, 0.1% SDS for 20 min at 65°C. The membranes were exposed to a phosphor screen (Molecular Dynamics, Sunnyvale, CA) for 4 hr, and the radioactivity in each band was quantified with a Storm 840 Phosphorlmager (Molecular Dynamics).

These experiments revealed that the CACl, CAC2 and CAC3 mRNAs are 1.1- kb, 2.0-kb and 2.9-kb in size, respectively. As previously reported (Woodbury et al., Curr Genet 14: 75-89 (1988); and Meurer et al., Plant Cell 8:1193-1207 (1996)), multiple accD RNAs accumulate; in developing siliques of Arabidopsis we detected two αccEJ-hybridizing transcripts, which are 2.3-kb and 1.5-kb in size. We termed these transcripts accD-A and accD-B, respectively. As deduced from the sequence of the Arabidopsis accD gene, both transcripts are of sufficient size to code for the β subunit of carboxyltransferase.

During silique development, the temporal accumulation of the CACl, CAC2, CAC3, accD-A and accD-B mRNAs is coordinated. At the beginning of development (1-2 days after flowering), the accumulation of these mRNAs is stable, then they transiently decline (3-4 days after flowering), before rising to maximum accumulation at 6-7 days after flowering. This peak in accumulation coincides with the maximal rate of seed oil accumulation. Subsequently, the accumulation of these mRNAs declines to about 1/10th of maximal levels by 10 days after flowering.

Example 2

This example describes the tissue distribution of the CACl, CAC2, CAC3 and accD mRNAs in Arabidopsis siliques at between 1 and 15 days after flowering (i.e., from pre-fertilization to the onset of dessication) using in situ hybridization.

Arabidopsis siliques (1 to 15 days after flowering) and flower buds were harvested and cut into 3-4 mm long pieces. Tissues were fixed, dehydrated, embedded and sectioned as previously described (Wang et al., Amer. J. Bot. 82: 1083 (1995); and Ke et al., Plant Physiol. 113: 357 (1997)). ³⁵S-labeled probes were transcribed from vectors containing the CACl, CAC2, CAC3 or accD cDNA (cloned in pBluescript SK). The labeled probes were hybridized to the tissue sections as described in Ke et al. (1997), supra. After hybridization, the tissue sections were coated with Kodak NTB2 emulsion, exposed for 2 to 4 days, and developed. Photographs were taken with an Orthopha microscope (Leitz, Wetzlar, Germany) using bright-field optics.

In situ hybridizations were repeated three times, using two sets of plant materials that had been independently processed, all with similar results. Control slides containing sections of siliques were hybridized with sense RNA probes transcribed from the vectors indicated above, and virtually no signal was detected in these slides.

CACl, CAC2, CAC3 and accD mRNAs accumulated evenly throughout the tissues of the silique at an early stage of development. Thus, at 1-3 days after flowering, these mRNAs were found in the silique wall, central septum, and ovules of the silique. Four days after flowering and later, accumulation of these mRNAs was greatly reduced in the tissues of the silique (silique walls, central septum and ovules) in which growth had ceased and oil did not accumulate. In contrast, as embryos developed within the ovules from globular to torpedo stage (4-7 days after flowering), the CACl, CAC2, CAC3 and accD mRNAs accumulated to ever-increasing levels within the embryos, reaching a peak at about seven days after flowering, when the embryos were at the elongated torpedo stage. Subsequently, the accumulation of all three mRNAs decreased, so that by 12 days after flowering, they were barely detectable. These developmentally induced changes in the accumulation of the CACl,

CAC2, CAC3 and accD mRNAs closely follow the pattern of growth and oil accumulation in the siliques. In particular, between 4 and 8 days after flowering, when a large number of oil bodies were deposited within the embryos and expanded in size as they filled with triacylglycerol, the accumulation of the ACC mRNAs reached maximal levels.

Example 3

This example describes the expression of a vector comprising a 1.1 kb Hin dffl-Pvtt II CACl promoter fragment fused in-frame to the B-glucuronidase (GUS) reporter gene in transformed plants.

A 1.1 kb Hin άlll-Pvu I CACl promoter fragment (-1048 to +32, relative to the ATG start codon) was fused in- frame to the GUS reporter gene in the plant transformation vector pBHOl (Clontech). The resulting plasmid carrying the chimeric gene, CACl -GUS, was transformed via Agrobacterium-mediated transformation into Arabidopsis. The resultant transgenic plants were selfed for three generations. Plants that were homozygous for the transgene were used to study the expression of CACl -GUS. The expression of CACl -GUS was examined by staining for GUS activity in planta. GUS activity was visualized by incubating seedlings for up to 18 hr at 37°C in a solution containing 5-bromo-4 chloro-3-indolyl β-D- glucuronic acid as described previously (Jefferson et al., EMBO J. 6: 3901-3907 (1987)) with modifications by De Block (Methods Cell Biol. 49: 153-163 (1995)). Following staining, chlorophyll was removed in the presence of 70% ethanol.

The staining for GUS activity revealed that the spatial and temporal patterns of GUS activity derived from the CACl-GUS closely mirrored that of CACl mRNA accumulation as determined by Northern blot and in situ hybridization analyses as set forth in Examples 1 and 2. In other words, CACl-GUS was highly expressed in growing and lipogenic tissues. Specifically, during silique development, CACl-GUS was initially active throughout the tissues of the silique, when the siliques were still expanding, but, later, as the embryo visibly developed and became the major lipogenic tissue in the silique, expression was concentrated within the embryo. The results indicate that the 1.1 kb Hin dlll-Pvw I promoter fragment from the CACl gene contains all of the genetic information required for the correct spatial and temporal pattern of CACl expression. Furthermore, this genetic information includes the means by which the expression of CACl is coordinated with the expression of the CAC2, CAC3 and accD genes.

Example 4

This example describes the use of promoter deletions of CACl-GUS in order to characterize further the CACl promoter.

A promoter deletion derivative of the CACl-GUS ransgene comprising -785 to +32 of the promoter, i.e., the 0.8 kb Msp l-Pvu II fragment (A-785 CACl -GUS), and a deletion derivative of CACl-Gus comprising -529 to +32 of the promoter, i.e., the 0.55 kb Hpa l-Pvu II fragment (A-529CAC1-GUS), were generated. CACl-GUS, Δ- 785CAC1-GUS and A-529CAC1 -GUS were separately transformed into Arabidopsis as described in Example 3. GUS activity was stained in these transgenic plants as in Example 3. The spatial and temporal pattern of GUS expression obtained from Δ- 785CAC1-GUS was indistinguishable from that obtained with CACl-GUS.

Therefore, the sequences that were deleted to construct A-785CAC1-GUS (situated between -1048 and -785) do not play a significant role in directing CACl expression in developing siliques and seeds. However, the spatial and temporal pattern of GUS expression obtained from A-529CAC1 -GUS was dramatically different from that obtained with CACl-GUS. The major changes in expression were evident during floral and silique development. During the development of the flowers and siliques, Δ -529CAC1 -GUS-deήyed GUS activity was greatly enhanced and occurred in tissues that do not normally accumulate high levels of CACl mRNA or express GUS from the CACl-GUS tτansgene, i.e., the stems, sepals, carpels, stamens, and silique walls and all of the internal tissues of the siliques. The degree of this enhancement was qualitatively evident during the GUS activity assays. The blue color was readily apparent within the first hour of the assay for A-529CAC1-GUS fransgenics, whereas the blue color was not apparent until six hours into the assay form CACl-GUS fransgenics. Whereas CACl-GUS-deήved GUS expression was constricted to the developing seeds of the siliques that were older than five days after flowering, GUS activity derived from A-529CAC1 -GUS was expressed in all of the tissues of the siliques throughout their development. These data indicate that sequences located between positions -785 and -529 of the CACl promoter are critical for the correct expression of the CACl gene. Furthermore, these sequences act in cis to suppress the expression of the CACl gene in inappropriate tissues and times of development.

Example 5

This example describes how the level of a monomeric ACC, a subunit of a heteromeric ACC, ACC enzymatic activity or malonyl CoA in a plant can be increased by increasing the expression of one or more ACC genes.

The level of a monomeric ACC, a subunit of a heteromeric ACC, ACC enzymatic activity or malonyl CoA generated in a cell of a plant can be increased, for example, by increasing the accumulation of a monomeric ACC or one or more subunits of a heteromeric ACC. This can be achieved by introducing into the genome of an organism copies of one or more ACC genes or cDNAs fused to novel expression regulatory sequences that express the gene(s) at higher levels than normal, such as the modified promoters provided herein. A copy of the ACC gene or cDNA is fused to upstream (5') and/or downstream (3') transcriptional or translational regulatory sequences and the chimeric gene is cloned into an appropriate transformation vector that carries a selectable marker gene and the vector is transformed into the organism of choice. Transformants are selected on the basis of the marker gene. Transformants are confirmed by Southern blot analysis of the DNA from putative transformants. Multiple copies of each novel ACC gene or combinations of novel ACC genes can be introduced into the genome of an organism. A copy of each gene is cloned into an appropriate transformation vector that carries a selectable marker gene and the vector is transformed into the organism of choice. Transformants are selected on the basis of the marker gene. Transformants are confirmed by Southern blot analysis of the DNA from putative transformants. In some cases, this single transformation event will introduce multiple copies of an ACC gene. Alternatively, multiple copies of an ACC gene are cloned into the transforming vector. Alternatively, an ACC gene is cloned into transformation vectors that carry different selectable marker genes and multiple transformations are carried out to introduce multiple copies of an ACC gene. In some cases, it is necessary to introduce a combination of ACC genes. This is achieved by cloning a combination of ACC genes into the same transformation vector or into different transformation vectors that carry different selectable marker genes.

Example 6

This example describes how the level of a monomeric ACC, a subunit of a heteromeric ACC, ACC enzymatic activity or malonyl CoA in a plant can be increased or decreased by using gene replacement.

The level of monomeric ACC, a subunit of heteromeric ACC, ACC enzymatic activity or malonyl CoA generated in a cell of a plant can be altered by altering the activity of a monomeric ACC or one or more subunits of a heteromeric ACC. This can be achieved by a gene replacement method via homologous recombination. In this method, the endogenous ACC gene is replaced by a mutagenized ACC gene. The mutagenized ACC gene codes for a subunit of an enzyme that is either more or less efficient in catalysis than the one encoded by the endogenous, replaced gene. The ACC gene is mutagenized by one or more nucleotide deletions, insertions, duplications or replacements and/or by the use of a modified promoter as described herein. The mutagenized gene is fused to a selectable marker gene and introduced into a cell. Homologous recombination events that may result in gene replacement are selected on the basis of the selectable marker gene. Gene replacements are confirmed by Southern blot analysis or PCR and DNA sequencing.

Example 7 This example describes how to decrease the level of a monomeric ACC, a subunit of a heteromeric ACC, ACC enzymatic activity or malonyl CoA in a plant by using co-suppression.

The level of a monomeric ACC, a subunit of a heteromeric ACC, ACC enzymatic activity or malonyl CoA generated in a cell of a plant can be decreased by decreasing the accumulation of a monomeric ACC or one or more of the subunits of a heteromeric ACC. This can be achieved by co-suppression. For example, the cDNA coding for a subunit of ACC is fused to upstream (5') and/or downstream (3') transcriptional or translational regulatory sequences, such as the suppressor element provided herein, and the chimeric gene is cloned into an appropriate transformation vector that carries a selectable marker gene and the vector is transformed into the organism of choice. Transformants are selected on the basis of the marker gene. Transformants are confirmed by Southern blot analysis of the DNA from putative transformants. Most of the transgenic organisms that will be derived from such experimentations will express the transgene. However, in a few cases, the transgene will co-suppress the expression of the endogenous ACC gene. To identify these co- suppressing plants, extracts from at least 100 transgenic plants will be analyzed for the enzymatic activity of ACC.

Example 8 This example describes how to increase the level of a monomeric ACC, a subunit of a heteromeric ACC, ACC enzymatic activity or malonyl CoA by overexpressing an ACC subunit in a model organism (i.e., Arabidopsis).

The full-length ACC cDNA is cloned into a plant expression vector such as pBHOl, down-stream of a modified promoter as provided herein. The resulting recombinant vector is transformed into Agrobacterium tumefaciens. The resulting strain is used to transform Arabidopsis plants by vacuum infiltration protocols. Namely, flower buds of Arabidopsis are dipped for 1-5 minutes into a culture of the Agrobacterium tumefaciens strain. Plants are allowed to set seed, which are collected. Seeds are germinated on agar plates containing 50-100 μg/ml kanamycin, and resistant, transformed seedlings that grow on this medium are transferred to soil. Between 10 and 50 independently transformed seedlings are collected and allowed to flower and set seed. This T2 generation of seed is homozygous for the transgene. Confirmation of the transgenic nature of the seed is undertaken by extracting DNA from the resulting T2 generation seedlings and performing Southern blot analysis using the ACC cDNA and the CaMV, 35S promoter as probes. Transgenic plants are tested for expression of the ACC transgene, for increased ACS activity and for increased accumulation of ACC transcripts or malonyl CoA.

Expression of the ACC transgene is carried out by analyzing the accumulation of the ACC mRNA and polypeptide. The ACC mRNA can be detected by Northern hybridization with the ACC cDNA, or by RNase protection assays using an ACC transgene-specific probe.

The ACC polypeptide is detected by Western blot analysis of total proteins separated by SDS-PAGE and probed with ACC-specific antibodies. ACC activity is determined by incubating an extract with labeled acetyl CoA and monitoring the production of labeled malonyl CoA. The accumulation of malonyl CoA is monitored by extracting seedlings with 10% trichloroacetic acid.

The resulting extract is subjected to High Pressure Liquid Chromatography, using a C-18 reverse phase column. The solvent for elution is KH₂PO₄, pH 5.5, in acetonitrile. Elution of malonylCoA is identified by co-elution with authentic malonyl CoA. Malonyl CoA concentration is determined based on absorbance at 254 nm.

Example 9

This example describes the use of a modified promoter of the CACl gene of Arabidopsis thaliana, wherein the promoter comprises nucleotides -529 to +32 relative to the translation start codon of CACl, operably linked to the coding sequence of ATP citrate lyase (ACL) to transform plants. The full-length ACL cDNA, operably linked to a deleted CACl promoter (nucleotides -529 to +32) in sense orientation, was cloned into a derivative of the pBI121 plasmid (Clonetech), which comprises the kanamycin resistance (kan-r) gene, using standard techniques (Sambrook et al., Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory, New York (1989)). Restriction endonucleases and DNA ligase (GibcoBRL, Grand Island, NY) were used with protocols suggested by the manufacturers. The resulting vectors were separately transformed into Agrobacterium tumefaciens. The resulting strain was used to transform Arabidopsis thaliana (ecotype Columbia) plants by vacuum infiltration protocols, based on the procedures described by Bechtold et al. (Methods Molec. Biol. 82: 259-266 (1993)). Briefly, flower buds of Arabidopsis were dipped for 1 min into a culture of the A. tumefaciens strain. Plants were allowed to set seed, which was then collected.

Transgenic plants were generated and selected on the basis of kanamycin resistance. For this selection, seeds were surface-sterilized by incubating them for 7 min in 50% (v/v) regular bleach (5.25 % sodium hypochlorite) and 0.02 % Triton X- 100 followed by rinsing them three times with sterile water. Seeds were sown in Petri plates containing MS selection medium (50 μg/ml kanamycin, 1 x Murashige and Skoog's salts (Sigma Chemical Co., St. Louis, MO), 1 % sucrose, 1 x Gamborg's vitamin (Sigma), 0.5 g/1 MES, pH 5.7, and 0.8 % purified agar (Becton Dickinson, Cockeysville, MD)). Approximately 10-14 days after sowing, kanamycin-resistant seedlings were transferred into sterile soil (Sunshine Mix, Sun Gro Horticulture, Bellevue, WA). Plants were grown at 23°C either under continuous light or under a photoperiod of 16 hrs illumination followed by 8 hrs of darkness. Plants were watered once a week with Nutriculture soluble fertilizer special blend 21-8-18 (Plant Marvel Laboratory, Chicago Heights, IL).

Each transgenic line from an individual transformed plant was considered to be an independent transformation event. In the following generations, seeds were harvested from transgenic lines individually and were further grown in MS selection medium to investigate the segregation of the kanamycin resistance trait. For each test of the segregation of kanamycin resistance, more than 30 seeds were used. A transgenic line was considered to be homozygous when there was only one copy of the transgene incorporated into its genome and all of the tested progeny seedlings (more than 50) were kanamycin resistant.

Transgenic plants have been grown through the T2 generation. Such plants exhibit an altered phenotype of very large leaves.

Example 10

This example describes the cloning and sequencing of the CAC3 genomic DNA.

An Arabidopsis genomic library, from the ecotype Landsberg erecta (Voytas et al., Genetics 126: 713 (1990)), cloned in the vector λFIX, was obtained from the Arabidopsis Biological Resource Center (Ohio State University, Columbus). Approximately 40,000 recombinant bacteriophage were screened by hybridization (Sambrook et al. (1989), supra) with the Arabidopsis EST cDNA clone GBGelό (Genbank accession #Z25579), which codes for the alpha-transcarboxylase subunit of the heteromeric acetyl-CoA carboxylase of Arabidopsis. Five of 11 hybridizing clones were plaque-purified and further analyzed Sambrook et al. (1989), supra). Restriction digests and Southern blot hybridization analyses using 5'- and 3 '-end specific probes from the GBGelό cDNA revealed that these five clones contained overlapping segments of the Arabidopsis genome, and that two of these clones contained both ends of the CAC3 gene; inserts from these phage were subcloned into pBluescript SK and sequenced.

In total, more than 6-kb of a contiguous stretch of Arabidopsis genome was sequenced. Comparison of this sequence to the full-length CAC3 cDNA sequence identified the structure of the CAC3 gene (see Fig. 3 in which lower case letters represent intronic sequences and upper case letters represent exonic sequences. The CAC3 gene is interrupted by 11 infrons that range from 73 to 203 nucleotides in length. The nucleotide sequences at the intron-exon-intron junctions follow characteristic patterns observed in other plant genes (Brown, Nucleic Acids Research 14: 9549-9559 (1989)); Ghislain et al., Plant Mol. Biol. 24: 835-851 (1994)). The exception to this rule is the 3 '-end of intron 3, which has the sequence, 5'-TCC^G. Southern blot analysis of Arabidopsis genomic DNA shows single hybridizing bands of the size predicted from the CAC3 sequence for Bam HI, Eco RI, Sal I, and Xho I digests. Furthermore, Eco RV digestion generates two hybridizing bands of different intensities, consistent with the CAC3 gene sequence, which contains a unique Eco RV site near the 3' end of the gene. These data indicate that the G4C3 gene occurs only once in the Arabidopsis genome.

All of the references cited herein, including patents, patent applications, and publications, are hereby incorporated in their entireties by reference.

While this invention has been described with an emphasis upon preferred embodiments, it will be apparent to those of ordinary skill in the art that variations in the preferred embodiments can be prepared and used and that the invention can be practiced otherwise than as specifically described herein. The present invention is intended to include such variations and alternative practices. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the invention as defined by the following claims.

Claims

WHAT IS CLAIMED IS:

1. An isolated or purified nucleic acid fragment consisting essentially of a modified promoter of the CACl gene of Arabidopsis thaliana, wherein said promoter comprises bases from about -1048 to about -1 relative to the translation start codon of said gene and is modified in the region from about -785 to about -529 relative to the translation start codon so as to inactivate a suppressor element present in said region.

2. An isolated or purified nucleic acid fragment consisting essentially of a modified promoter of the CAC2 gene of Arabidopsis thaliana, wherein said promoter comprises bases from about -1412 to about -1 relative to the translation start codon of said gene and is modified in the region from about -880 to about -586 relative to the translation start codon so as to inactivate a suppressor element present in said region.

3. An isolated or purified nucleic acid fragment consisting essentially of a modified promoter of the CAC3 gene of Arabidopsis thaliana, wherein said promoter comprises bases from about -1561 to about -1 relative to the translation start codon of said gene and is modified in the region from about -422 to about -1,000 relative to the translation start codon so as to inactivate a suppressor element present in said region.

4. The isolated or purified nucleic acid fragment of any of claims 1-3, wherein said promoter is modified in said region by a deletion, insertion or substitution, wherein said deletion, insertion or substitution is of sufficient size and appropriate position to effect inactivation of a suppressorelement present in said region.

5. An isolated or purified nucleic acid fragment consisting essentially of a suppressor element of the promoter of the CACl gene of Arabidopsis thaliana, wherein said suppressor element comprises bases from about -785 to about -529 relative to the translation start codon of said gene.

6. An isolated or purified nucleic acid fragment consisting essentially of a suppressor element of the promoter of the CAC2 gene of Arabidopsis thaliana, wherein said suppressor element comprises bases from about -880 to about -586 relative to the translation start codon of said gene.

7. An isolated or purified nucleic acid fragment consisting essentially of a suppressor element of the promoter of the CAC3 gene of Arabidopsis thaliana, wherein said suppressor element comprises bases from about -422 to about -1,000 relative to the translation start codon of said gene.

8. A recombinant vector comprising an isolated or purified nucleic acid fragment of any of claims 1-4 operably linked to a coding sequence to be expressed in a cell.

9. A host cell comprising a recombinant vector of claim 8.

10. A method of regulating the expression of a coding sequence in a cell, which method comprises contacting said cell, with a vector comprising an isolated or purified nucleic acid fragment of any of claims 1-4 operably linked to the coding sequence, wherein said nucleic acid fragment regulates the expression of the coding sequence in the cell.

11. A method of regulating the expression of a coding sequence in a cell, which method comprises randomly inserting an isolated or purified nucleic acid fragment of any of claims 1-7 into the genome of the cell, whereupon a given random insertion, the expression of a coding sequence in the cell is regulated by said nucleic acid fragment.

12. A method of suppressing the expression of a coding sequence in a cell, which method comprises randomly inserting the isolated or purified nucleic acid fragment of any of claims 5-7 into the genome of the cell, whereupon a given random insertion, the expression of a coding sequence in the cell is suppressed by said nucleic acid fragment.

13. A method of suppressing the expression of a coding sequence in a cell, which method comprises nonrandomly inserting the isolated or purified nucleic acid fragment of any of claims 5-7 into the genome of the cell, whereupon targeted insertion, the expression of the coding sequence in the cell is suppressed by said nucleic acid fragment.

14. A plant cell in which the expression of a coding sequence in the plant cell has been regulated in accordance with the method of claim 10 or 11.

15. A plant tissue, a plant organ or a plant comprising the plant cell of claim 14.

16. A plant cell in which the expression of a coding sequence in the nucleus of the plant cell has been suppressed in accordance with the method of claim 12 or 13.

17. A plant tissue, a plant organ or a plant comprising the plant cell of claim 16.

18. An isolated or purified nucleic acid molecule comprising the genomic DNA sequence of a carboxyltransferase ╬▒ subunit of a plant acetyl CoA carboxylase or a continuous fragment thereof comprising at least about 20 nucleotides, wherein the continuous fragment is not derived entirely from an exon of the genomic DNA sequence.

19. The isolated or purified nucleic acid molecule of claim 18, wherein said nucleic acid molecule is isolated from Arabidopsis.

20. The isolated or purified nucleic acid molecule of claim 18, wherein said nucleic acid molecule comprises SEQ ID NO: 3, a continuous fragment thereof comprising at least about 20 nucleotides, wherein the continuous fragment is not derived entirely from an exon of the genomic DNA sequence, or a nucleic acid molecule comprising a genomic DNA sequence of a carboxyltransferase ╬▒ subunit of a plant acetyl CoA carboxylase that hybridizes to either one of the foregoing under stringent conditions.

21. An isolated or purified nucleic acid molecule encoding a modified carboxyltransferase ╬▒ subunit of a plant acetyl CoA carboxylase, which comprises one or more insertions, deletions and/or substitutions, wherein the modified subunit encoded by said isolated or purified nucleic acid molecule does not differ functionally from the corresponding unmodified subunit, or a continuous fragment thereof comprising at least about 20 nucleotides.

22. The isolated or purified nucleic acid molecule of claim 21 , wherein the corresponding unmodified subunit SEQ ID NO: 3.

23. The isolated or purified nucleic acid molecule of claim 22, wherein the modified subunit encoded by the isolated or purified nucleic acid molecule, together with the remaining unmodified subunits of a plant acetyl CoA carboxylase, converts acetyl CoA to malonyl CoA at least about 90% as well as the plant acetyl CoA carboxylase comprising the subunit comprising SEQ ID NO: 3 as determined by in vitro assay using labeled acetyl CoA.

24. A vector comprising a nucleic acid molecule of any of claims 18-23.

25. A host cell comprising the vector of claim 24.

26. A method of altering the level of a subunit of acetyl CoA carboxylase in a plant cell, a plant tissue, a plant organ, or a plant, which method comprises contacting said plant cell, plant tissue, plant organ or plant with a vector comprising a gene encoding a subunit of acetyl CoA carboxylase selected from the group consisting of biotin carboxyl carrier, biotin carboxylase, carboxyltransferase ╬▒, and carboxyltransferase ╬▓, wherein said gene comprises a modified promoter of any of claims 1-4, wherein said vector increases or decreases the level of said subunit in said plant cell, plant tissue, plant organ or plant.

27. The method of claim 26, wherein the alteration of the level of said subunit of acetyl CoA carboxylase results in an alteration of the level of acetyl CoA carboxylase enzymatic activity.

28. The method of claim 26, wherein the alteration of the level of said subunit results in an alteration of the level of malonyl CoA.

29. A plant cell, a plant tissue, a plant organ or a plant in which the level of a subunit of acetyl CoA carboxylase has been altered in accordance with the method of claim 26.

30. A plant cell, a plant tissue, a plant organ or a plant in which the level of acetyl CoA carboxylase enzymatic activity has been altered in accordance with the method of claim 27.

31. A plant cell, a plant tissue, a plant organ or a plant in which the level of malonyl CoA has been altered in accordance with the method of claim 28.