# Patent

## Title
Materials and methods for the alteration of enzyme and acetyl CoA levels in plants

## Abstract

The present invention provides nucleic acid and amino acid sequences of acetyl CoA synthetase (ACS), plastidic pyruvate dehydrogenase (pPDH), ATP citrate lyase (ACL), Arabidopsis pyruvate decarboxylase (PDC), and Arabidopsis aldehyde dehydrogenase (ALDH), specifically ALDH-2 and ALDH-4. The present invention also provides a recombinant vector comprising a nucleic acid sequence encoding one of the aforementioned enzymes, an antisense sequence thereto or a ribozyme therefor, a cell transformed with such a vector, antibodies to the enzymes, a plant cell, a plant tissue, a plant organ or a plant in which the level of an enzyme has been altered, and a method of producing such a plant cell, plant tissue, plant organ or plant. Desirably, alteration of the level of enzyme results in an alteration of the level of acetyl CoA in the plant cell, plant tissue, plant organ or plant. In addition, the present invention provides a recombinant vector comprising an antisense sequence of a nucleic acid sequence encoding pyruvate decarboxylase (PDC), the E1α subunit of pPDH, the E1β subunit of pPDH, the E2 subunit of pPDH, mitochondrial pyruvate dehydrogenase (mtPDH) or aldehyde dehydrogenase (ALDH) or a ribozyme that can cleave an RNA molecule encoding PDC, E1α pPDH, E1β pPDH, E2 pPDH, mtPDH or ALDH.

## Background
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
This patent application is a divisional of copending U.S. patent application Ser. No. 09/344,882, filed Jun. 25, 1999, which claims the benefit of U.S. Provisional Patent Application No. 60/090,717, filed Jun. 26, 1998.


STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
This invention was made, in part, with funding from the National Science Foundation under Grant No. IBN-9696154 and from the Department of Energy to the Consortium for Plant Biotechnology Research via the Prime Agreement No. DE-FC05-92OR22072. Therefore, the United States of America may have certain rights in the invention.


TECHNICAL FIELD OF THE INVENTION
The present invention relates to nucleic acid and amino acid sequences of acetyl CoA synthetase (ACS), plastidic pyruvate dehydrogenase (pPDH), ATP citrate lyase (ACL), pyruvate decarboxylase (PDC) from Arabidopsis, and aldehyde dehydrogenase (ALDH) from Arabidopsis. The present invention also relates to a recombinant vector comprising (i) a nucleic acid sequence encoding an aforementioned enzyme, (ii) an antisense sequence thereto or (iii) a ribozyme therefor, a cell transformed with such a vector, antibodies to the enzymes, a plant cell, a plant tissue, a plant organ or a plant in which the level of an enzyme or acetyl CoA, or the capacity to produce acetyl CoA, has been altered, and a method of producing such a plant cell, plant tissue, plant organ or plant. In addition, the present invention relates to a recombinant vector comprising (i) an antisense sequence to a nucleic acid sequence encoding PDC, the E1α subunit of pPDH, the E1β subunit of pPDH, the E2 subunit of pPDH, mitochondrial pyruvate dehydrogenase (mtPDH) or ALDH or (ii) a ribozyme that can cleave an RNA molecule encoding PDC, E1α pPDH, E1β pPDH, E2 pPDH, mtPDH or ALDH.
BACKGROUND OF THE INVENTION
ACS and pPDH are two enzymes that are responsible for the generation of acetyl CoA in the plastids, e.g., chloroplasts, of plants. ACS generates acetyl CoA as follows:

acetate+ATP+CoASH→acetyl-CoA+AMP+PPi,

wherein ATP represents adenine triphosphate, CoASH represents coenzyme A, acetyl-CoA represents acetyl coenzyme A, AMP represents adenine monophosphate, and PPi represents inorganic pyrophosphate, and wherein the acetate includes that which results from the conversion of acetaldehyde and NAD+ to acetate and NADH, wherein the acetaldehyde, in turn, results from the breakdown of pyruvate, which releases CO2. pPDH generates acetyl CoA as follows:

pyruvate+CoASH+NAD+→acetyl-CoA+CO2+NADH,

wherein NAD+ represents nicotinamide adenine dinucleotide and NADH represents the reduced form of NAD+ and wherein the pyruvate results from glycolysis. Glycolysis involves the conversion of sugar phosphates, which have been produced from starch, photosynthesis or the importation of triose and hexose phosphates from the cytosol, to pyruvate.

Various studies of relative activity of enzymes in embryos and leaves of plants, such as spinach, castor bean, barley and Brassica have been conducted (see, Kang and Rawsthorne, Plant J. 6: 795-805 (1994); Miernyk and Dennis, J. Exper. Bot. 34: 712-718 (1983); Smith et al., Plant Physiol. 98: 1233-1238 (1992); Liedvogel and Bauerle, Planta 169: 481-489 (1986); Murphy and Leech, FEBS Letter 77: 164-168 (1977); Roughan et al., Biochem. J. 158: 593-601 (1976); Roughan et al., Biochem. J. 184: 565-569 (1978); Roughan et al., Biochem. J. 184: 193-202 (1979); Springer and Heise, Planta 177: 417-421 (1989); Schulze-Siebert and Shultz, Plant Physiol. 84: 1233-1237 (1987); and Heintze et al., Plant Physiol. 93: 1121-1127 (1990)). Such studies suggest that acetate is the preferred substrate for fatty acid synthesis in chloroplasts, while pyruvate is the preferred substrate for fatty acid synthesis in plastids in embryos.
The acetyl CoA so produced is then involved in fatty acid biosynthesis, i.e., the synthesis of the basic building blocks of membrane lipids, fats and waxes. A similar reaction is effected by mtPDH in the mitochondrion.
ACS is exclusively found in the plastids of plants and is strongly regulated by light (Sauer and Heise, Z. Naturforsch 38c: 399-404 (1983)). The amount of ACS is fairly constant between spinach, pea and amaranthus chloroplasts; there is about 20% more in corn chloroplasts. Given that the partially purified enzyme is completely DTT-dependent suggests that its activity in vivo may be regulated by the ferredoxin/thioredoxin system (Zeiher and Randall, Plant Physiol. 96: 382-389 (1991)). There is some potential for weak feedback inhibition by acetyl CoA. The enzyme also has a high pH requirement, along with a dependency on a high ATP(Mg2+-ATP)/ADP ratio (Sauer and Heise (1983), supra). The ACS reaction should be substrate saturated because Km values for acetate are between 0.02 and 0.10 mM in spinach (Sauer and Heise (1983), supra; Zeiher and Randall (1991), supra; and Treede and Heise, Z. Naturforsch 40c: 496-502 (1985)), peas (Treede and Heise (1985), supra), amaranthus (Roughan and Ohlrogge, Anal. Biochem. 216: 77-82 (1994)) and potatoes (Huang and Stumpf, Arch Biochem. Biophys. 140: 158-173 (1970)), whereas the concentration of cellular acetate is estimated to be about 1.0 mM (Kuhn et al., Arch Biochem. Biophys. 209: 441-450 (1981)).
The pPDH appears to have the same general structure as mtPDH, being composed of a pyruvate dehydrogenase component (E1α and E1β), a transacetylase component (E2), and dehydrolipoamide dehydrogenase (E3) subunits. The molecular weight of pPDH and its cofactor requirements are also similar to mtPDH, although affinities for NAD+ and TPP vary somewhat (Camp and Randall, Plant Physiol. 77: 571-577 (1985); Miernyk et al. (1983), supra; and Conner et al., Planta 200: 195-202 (1996)). pPDH, which is less sensitive to acetyl CoA than mtPDH, has an optimal pH of about 8.0 and requires about 10 mM Mg2+ for maximal activity. While the activity of mtPDH is controlled by a sophisticated kinase/phosphatase system, which phosphorylates and thereby inactivates the E1α subunit, pPDH is not subject to such regulation. However, pPDH is strongly regulated by the NADH/NAD+ ratio and is moderately regulated by light. Regulation by ATP, NADPH, fatty acyl CoAs and glycolytic intermediates is minor (Camp et al., Biochim. Biophys. Acta 933: 269-275 (1988); and Qi et al., J. Exp. Bot. 47: 1889-1896 (1996)).
PDH activity varies from one tissue to the next with mtPDH activity varying 15-fold and pPDH activity varying 6-fold (Lernmark and Gardestrom, Plant Physiol. 106: 1633-1638 (1994)). The ratio of pPDH/mtPDH also varies between plants, with 6.5 times more activity in the chloroplasts than in the mitochondria of wheat leaves to 6.7 times more activity in the mitochondria than in the chloroplasts of peas. Although chloroplasts have proportionally less PDH activity than mitochondria in pea as compared to wheat, the chloroplasts have nearly as much PDH as mitochondria in absolute terms.
ACL is an enzyme that is responsible for the generation of acetyl CoA. ACL generates acetyl CoA as follows:

citrate+ATP+CoASH→acetyl-CoA+oxaloacetate+ADP+Pi,

wherein ADP represents adenosine diphosphate and Pi represents orthophosphate and wherein the citrate is that which is generated in the TCA cycle in the mitochondrion. The activity of ACL has been found to correlate with lipid accumulation in developing seeds of Brassica napus L. (Ratledge et al., Lipids 32(1): 7-12 (1997)) and in the supernatant of a developing soybean (Glycine max L. Merr., var. Harosoy 63) cotyledon homogenate (Nelson et al., Plant Physiol. 55: 69-72 (1975)). ACL also has been found in crude extracts from the endosperm tissue of germinating castor bean (Ricinus communis cv. Hale) and has been found to be maximally active in 4-5-day old seedlings (Fritsch et al., Plant Physiol. 63: 687-691 (1979)).

PDC is a cytosolic enzyme that is responsible for the generation of acetaldehyde from pyruvate. PDC generates acetaldehyde from pyruvate as follows:

pyruvate→acetaldehyde+CO2.

The acetaldehyde so produced can be acted upon by ALDH.

ALDH is responsible for the generation of acetate from acetaldehyde. ALDH generates acetate from acetaldehyde as follows:

acetaldehyde+NAD++H2O→acetate+NADH++H+.

The acetate so produced can then enter the plastids, where it can be converted to acetyl CoA through the action of ACS.

ACH is an enzyme that is known to exist in yeast and is believed to exist in the mitochondria of plants. ACH is believed to generate acetate from acetyl CoA pools present in the mitochondria. The acetate so produced is then believed to be released from the mitochondrion into the cytosol. The cytosolic acetate can then enter the plastids, wherein it can be converted to acetyl CoA through the action of ACS.
Acetyl CoA is the common precursor of a large number of phytochemicals, which have widely varied biological functions and which represent renewable, energy-rich products of agriculture (e.g., fats, oils, waxes, isoprenoids and bioplastics (e.g., polyhydroxybutyrate) or which affect agricultural production (e.g., flavonoids, stilbenoids, isoprenoids and malonyl derivatives) (Goodwin and Mercer, Introduction to Plant Biochemistry, 2nd ed., Pergamon Press, New York (1988)). These phytochemicals are synthesized either by the carboxylation or sequential condensation of acetyl CoA.
Carboxylation of acetyl CoA (via the intermediate malonyl CoA) leads to the biosynthesis of fatty acids (e.g., membranes, oils, cuticle, suberin and cutin), flavonoids (e.g., pigments, phytoalexins and plant protection), stilbenoids (e.g., plant protection and pharmaceuticals), acridones, malonic acid, and a variety of malonyl derivatives (aminocyclopropane carboxylic acids, D-amino acids, flavonoids and pesticides). Fatty acids are the building blocks of all cellular membranes. In addition, fatty acids are utilized in developmentally regulated processes in the biogenesis of seed oils, cuticle, cutin and suberin. Most seed oils are triacylglycerols. Flavonoids are a group of water-soluble phenolic compounds that have a wide range of biological activities as pigments and they accumulate in responses of plants to biotic and abiotic stresses (e.g., drought, fungal and bacterial pathogens, and salt stress). Stilbenoids are thought to play a role in plant defense mechanisms. The acridones are a class of alkaloids that have a wide spectrum of antimicrobial, antimolluscosidal and antiviral activities. Numerous malonyl derivatives exist in plants, including those of D-amino acids, flavonoids and xenobiotics, such as pesticides. The malonation of aminocyclopropanecarboxylic acid, which is the precursor of ethylene, may influence the generation of the hormone ethylene.
The condensation of acetyl CoA (via the intermediates acetoacetyl CoA and HMG CoA) leads to the biosynthesis of isoprenoids. Examples of isoprenoids include sterols, phytoalexins, abscisic acid, gibberellins, phytoene, β-carotene, phytol, natural rubber, plant protection and pharmaceuticals. Isoprenoids are also significant constituents of many essential oils and fragrances.
In addition, and of great excitement to the biotechnology industry, acetoacetyl CoA is the precursor for the production of a potentially new agricultural product from transgenic plants, namely polyhydroxybutyrate (PHB, a type of bioplastic). Research to date indicates that the production of transgenic bioplastics may be limited by the supply of acetyl CoA (Nawrath et al., PNAS USA 91: 12760-12764 (1994)).
The pathways that utilize acetyl CoA as a precursor are spatially and temporally compartmentalized. Fatty acids and sterols are synthesized by all cells for membrane biogenesis. The accumulation of most of the other acetyl CoA-derived phytochemicals is highly cell-specific and occurs in specific subcellular compartments at particular stages of development or in response to particular environmental signals. For example, acetyl CoA is required in plastids for de novo fatty acid synthesis, which produces 18-carbon fatty acids. The elongation of 18-carbon fatty acids to fatty acids of 20 carbons and longer requires a cytosolic acetyl CoA pool. Acetyl CoA is also required in the cytosol for the biosynthesis of isoprenoids, flavonoids, and several, if not all, of the malonated derivatives. In addition, fatty acid synthesis in the plastid should be maximal during triacylglycerol deposition in oil seed as well as during times of maximum membrane formation, such as during the conversion of proplastids to chloroplasts.
Therefore, in view of the above, there remains a need for materials and methods to alter the level of enzymes involved in acetyl CoA production and, consequently, acetyl CoA levels in plants. Accordingly, it is an object of the present invention to provide such materials and methods. These and other objects and advantages of the present invention, as well as additional inventive features, will become apparent to one of ordinary skill in the art from the following description.
BRIEF SUMMARY OF THE INVENTION
In one embodiment, the present invention provides isolated or purified nucleic acid molecules. One isolated or purified nucleic acid molecule encodes a plant plastidic ACS, such as that which is isolated from Arabidopsis, and a continuous fragment thereof comprising at least about 20 nucleotides. Preferably, the ACS-encoding nucleic acid molecule is (i) DNA and comprises SEQ ID NO: 1 or a sequence that encodes SEQ ID NO: 2, (ii) RNA and comprises a sequence encoded by SEQ ID NO: 1 or a sequence that encodes SEQ ID NO: 2, or (iii) a nucleic acid molecule that hybridizes to either one of the foregoing under stringent conditions. Also provided is an isolated or purified nucleic acid molecule encoding a modified ACS and a continuous fragment thereof comprising at least about 20 nucleotides.
Also in this regard, the present invention further provides an isolated or purified nucleic acid molecule encoding the E3 subunit of a plant pPDH (E3 pPDH), such as that which is isolated from Arabidopsis, and a continuous fragment thereof comprising at least about 20 nucleotides. Preferably, the E3 pPDH-encoding nucleic acid molecule is (i) DNA and comprises SEQ ID NO: 27 (E3-1 pPDH) or SEQ ID NO: 29 (E3-2 pPDH) or a sequence that encodes SEQ ID NO: 28 (E3-1 pPDH) or SEQ ID NO: 30 (E3-2 pPDH), (ii) RNA and comprises a sequence encoded by SEQ ID NO: 27 or SEQ ID NO: 29 or a sequence that encodes SEQ ID NO: 28 or SEQ ID NO: 30, or (iii) a nucleic acid molecule that hybridizes to either one of the foregoing under stringent conditions. Also provided is an isolated or purified nucleic acid molecule encoding a modified E3 subunit of pPDH and a continuous fragment thereof comprising at least about 20 nucleotides.
Another isolated or purified nucleic acid molecule encodes the A subunit of a plant ACL (ACL-A), such as that which is isolated from Arabidopsis, and a continuous fragment thereof comprising at least about 20 nucleotides. Preferably, the ACL-A-encoding nucleic acid molecule is (i) DNA and comprises SEQ ID NO: 7 or a sequence that encodes SEQ ID NO: 8, (ii) RNA and comprises a sequence encoded by SEQ ID NO: 7 or a sequence that encodes SEQ ID NO: 8 or (iii) a nucleic acid molecule that hybridizes to either one of the foregoing under stringent conditions. Also provided is an isolated or purified nucleic acid molecule encoding a modified A subunit of ACL and a continuous fragment thereof comprising at least about 20 nucleotides.
In this regard, the present invention further provides an isolated or purified nucleic acid molecule encoding the B subunit of a plant ACL (ACL-B), such as that which is isolated from Arabidopsis, and a continuous fragment thereof comprising at least about 20 nucleotides. Preferably, the ACL-B-encoding nucleic acid molecule is (i) DNA and comprises SEQ ID NO: 9 (ACL-B1) or SEQ ID NO: 11 (ACL-B2) or a sequence that encodes SEQ ID NO: 10 (ACL-B1) or SEQ ID NO: 12 (ACL-B2), (ii) RNA and comprises a sequence encoded by SEQ ID NO: 9 or SEQ ID NO: 11 or a sequence that encodes SEQ ID NO: 10 or SEQ ID NO: 12, or (iii) a nucleic acid molecule that hybridizes to either one of the foregoing under stringent conditions. Also provided is an isolated or purified nucleic acid molecule encoding a modified B subunit of ACL and a continuous fragment thereof comprising at least about 20 nucleotides.
An isolated and purified nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 15 or SEQ ID NO: 17 or encoding the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 18 is also provided by the present invention.
Likewise, an isolated and purified nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 21 or encoding the amino acid sequence of SEQ ID NO: 22 or a continuous fragment of either of the foregoing comprising at least about 20 nucleotides or a nucleic acid molecule that hybridizes to any of the foregoing under stringent conditions.
Similarly, an isolated and purified nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 25 or encoding the amino acid sequence of SEQ ID NO: 26 or a continuous fragment of either of the foregoing comprising at least about 20 nucleotides or a nucleic acid molecule that hybridizes to any of the foregoing under stringent conditions.
In another embodiment, the present invention also provides a vector comprising a nucleic acid molecule as described above, a host cell comprising such a vector, and a polypeptide produced by such a host cell. Also provided are vectors comprising or encoding an antisense sequence that hybridizes to or a ribozyme that cleaves an RNA molecule encoding plastidic ACS, the E1α subunit of pPDH, the E1β subunit of pPDH, the E2 subunit of pPDH, the E3 subunit of pPDH, the A subunit of ACL, the B subunit of ACL, PDC, ACH, mtPDH or ALDH, and a host cell comprising such a vector. In addition, antisense molecules, ribozymes and antibodies are provided.
In yet another embodiment, the present invention provides a method of altering the level of an enzyme 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 nucleic acid molecule selected from the group consisting of (i) a gene encoding an enzyme or, if the enzyme is comprised of subunits, a subunit of an enzyme selected from the group consisting of plastidic ACS, pPDH, ACL, pyruvate decarboxylase, acetyl CoA hydrolase, mitochondrial pyruvate dehydrogenase and aldehyde dehydrogenase, (ii) a nucleic acid molecule comprising or encoding an antisense molecule to an RNA molecule transcribed from a gene of (i), and (iii) a nucleic acid molecule comprising or encoding a ribozyme to an RNA molecule transcribed from a gene of (i). The vector comprising a nucleic acid molecule of (i) increases or decreases the level of an enzyme in the plant cell, plant tissue, plant organ or plant, whereas the vector comprising or encoding a nucleic acid molecule of (ii) or (iii) decreases the level of an enzyme in the plant cell, plant tissue, plant organ or plant. Preferably, the alteration of the enzyme results in an alteration of the level of acetyl 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 acetyl CoA has been altered in accordance with the method.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a description of cDNA [SEQ ID NO: 1] and amino acid [SEQ ID NO: 2] sequences for ACS.
FIG. 2 is a description of cDNA [SEQ ID NO: 3] and amino acid [SEQ ID NO: 4] sequences for the E1α subunit of pPDH.
FIG. 3 is a description of cDNA [SEQ ID NO: 5] and amino acid [SEQ ID NO: 6] sequences for the E1β-1 subunit of pPDH.
FIG. 4 is a description of cDNA [SEQ ID NO: 7] and amino acid [SEQ ID NO: 8] sequences for the A subunit of ACL.
FIG. 5 is a description of cDNA [SEQ ID NO: 9] and amino acid [SEQ ID NO: 10] sequences for the B-1 subunit of ACL.
FIG. 6 is a description of cDNA [SEQ ID NO: 11] and amino acid [SEQ ID NO: 12] sequences for the B-2 subunit of ACL.
FIG. 7 is a description of cDNA [SEQ ID NO: 13] and amino acid [SEQ ID NO: 14] sequences for the E1β-2 subunit of pPDH.
FIG. 8A is a description of the cDNA sequence [SEQ ID NO: 15] of PDC-1 from Arabidopsis. 
FIG. 8B is a description of the amino acid sequence [SEQ ID NO: 16] of PDC-1 from Arabidopsis. 
FIG. 9A is a description of the cDNA sequence [SEQ ID NO: 17] of PDC-2 from Arabidopsis. 
FIG. 9B is a description of the amino acid sequence [SEQ ID NO: 18] of PDC-2 from Arabidopsis. 
FIG. 10A is a description of the cDNA sequence [SEQ ID NO: 19] of ALDH-1 from Arabidopsis. 
FIG. 10B is a description of the amino acid sequence [SEQ ID NO: 20] of ALDH-1 from Arabidopsis. 
FIG. 11A is a description of the cDNA sequence [SEQ ID NO: 21] of ALDH-2 from Arabidopsis. 
FIG. 11B is a description of the amino acid sequence [SEQ ID NO: 22] of ALDH-2 from Arabidopsis. 
FIG. 12A is a description of the cDNA sequence [SEQ ID NO: 23] of ALDH-3 from Arabidopsis. 
FIG. 12B is a description of the amino acid sequence [SEQ ID NO: 24] of ALDH-3 from Arabidopsis. 
FIG. 13 is a description of cDNA [SEQ ID NO: 25] and amino acid [SEQ ID NO: 26] sequences for ALDH-4 from Arabidopsis. 
FIG. 14 is a description of cDNA [SEQ ID NO: 27] and amino acid [SEQ ID NO: 28] sequences for the E3-1 subunit of pPDH from Arabidopsis. 
FIG. 15 is a description of cDNA [SEQ ID NO: 29] and amino acid [SEQ ID NO: 30] sequences for the E3-2 subunit of pPDH from Arabidopsis.



DETAILED DESCRIPTION OF THE INVENTION
In one embodiment, the present invention provides isolated or purified nucleic acid molecules. 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.” “Nucleic acid molecules” is intended to encompass a polymer of DNA or RNA, i.e., a polynucleotide, which can be single-stranded or double-stranded and which can contain non-natural or altered nucleotides.
One isolated or purified nucleic acid molecule encodes a plant ACS, such as that which is isolated from Arabidopsis, and a continuous fragment thereof comprising at least about 20 nucleotides. Preferably, the ACS-encoding nucleic acid molecule is (i) DNA and comprises SEQ ID NO: 1 or a sequence that encodes SEQ ID NO: 2, (ii) RNA and comprises a sequence encoded by SEQ ID NO: 1 or a sequence that encodes SEQ ID NO: 2, or (iii) a nucleic acid molecule that hybridizes to either of the foregoing under stringent conditions. Also provided is an isolated or purified nucleic acid molecule encoding a modified ACS, which comprises one or more insertions, deletions and/or substitutions, and a continuous fragment thereof comprising at least about 20 nucleotides. Desirably, the modified ACS does not differ functionally from the corresponding unmodified ACS, such as that comprising SEQ ID NO: 2. Preferably, the modified ACS converts acetate to acetyl-CoA at least about 50%, more preferably at least about 75%, and most preferably at least about 90% as well as the corresponding unmodified ACS, such as that comprising SEQ ID NO: 2, as determined by in vitro assay using labeled acetate. Use of the word “labeled” herein is intended to mean any means of detection, such as a radioactive isotope.
The cDNA encoding ACS from Arabidopsis encodes a 76.7 kDa protein. The ACS of Arabidopsis is similar to that of E. coli and yeast, which contains two isoforms. Southern blot analysis of Arabidopsis DNA indicates that ACS is a single-copy gene. The ACS sequence from Arabidopsis is 56% similar and 48% identical to the ACS sequence of yeast and 66% similar and 58% identical to the ACS sequence of Arabidopsis. The sequence for ACS is available from GenBank as accession no. AF036618.
Another isolated or purified nucleic acid molecule provided by the present invention encodes the E1α subunit of a plant pPDH (E1α pPDH), such as that which is isolated from Arabidopsis, and a continuous fragment thereof comprising at least about 20 nucleotides (see, also, Johnston et al., BBA 1321: 200-206 (1997)). Preferably, the E1α pPDH-encoding nucleic acid molecule is (i) DNA and comprises SEQ ID NO: 3 or a sequence that encodes SEQ ID NO: 4, (ii) RNA and comprises a sequence encoded by SEQ ID NO: 3 or a sequence that encodes SEQ ID NO: 4, or (iii) a nucleic acid molecule that hybridizes to either one of the foregoing under stringent conditions. Also provided is an isolated or purified nucleic acid molecule encoding a modified E1α pPDH subunit, which comprises one or more insertions, deletions and/or substitutions, and a continuous fragment thereof comprising at least about 20 nucleotides. Desirably, the modified E1α pPDH subunit does not differ functionally from the corresponding unmodified E1α pDPH, such as that comprising SEQ ID NO: 4. Preferably, the modified E1α subunit of a plant pPDH, together with the remaining unmodified subunits of pPDH, does not differ functionally from a corresponding unmodified pPDH. Preferably, the modified pPDH converts pyruvate to acetyl-CoA at least about 50%, more preferably at least about 75%, most preferably at least about 90% as well as the corresponding unmodified pPDH, such as that comprising the E1α subunit comprising SEQ ID NO: 4, as determined by in vitro assay in the presence of labeled pyruvate.
The cDNA encoding the E1α subunit of pPDH from Arabidopsis encodes a 47 kDa protein. The E1α subunit is similar to that of Porphyra purpurea chloroplasts. Southern blot analysis of Arabidopsis DNA indicates that the E1α subunit is a single copy gene. The E1α subunit sequence from Arabidopsis is 61% identical to the E1α subunit of Porphyra purpurea E1α subunit sequence.
In this regard, the present invention further provides an isolated or purified nucleic acid molecule encoding the E1β a subunit of a plant pPDH (EE1β pPDH), such as that which is isolated from Arabidopsis, and a continuous fragment thereof comprising at least about 20 nucleotides (see also Johnston et al. (1997), supra). Preferably, the E1β pPDH-encoding nucleic acid molecule is (i) DNA and comprises SEQ ID NO: 5 (E1β-1 pPDH) or SEQ ID NO: 13 (E1β-2 pPDH) or a sequence that encodes SEQ ID NO: 6 or SEQ ID NO: 14, (ii) RNA and comprises a sequence encoded by SEQ ID NO: 5 or SEQ ID NO: 13 or a sequence that encodes SEQ ID NO: 6 or SEQ ID NO: 14, or (iii) a nucleic acid molecule that hybridizes to either one of the foregoing under stringent conditions. Also provided is an isolated or purified nucleic acid molecule encoding a modified E1β subunit of pPDH, which comprises one or more insertions, deletions and/or substitutions, and a continuous fragment thereof comprising at least about 20 nucleotides. Desirably, the modified E1β pPDH subunit does not differ functionally from the corresponding unmodified E1β pPDH subunit, such as that comprising SEQ ID NO: 6 or SEQ ID NO: 14. Preferably, the modified E1β subunit of a plant pPDH, together with the remaining unmodified subunits of pPDH, does not differ functionally from a corresponding unmodified pPDH. Preferably, the modified pPDH converts pyruvate to acetyl-CoA at least about 50%, more preferably at least about 75%, most preferably at least about 90% as well as the corresponding unmodified pPDH, such as that comprising the E1β subunit comprising SEQ ID NO: 6 or SEQ ID NO: 14, as determined by in vitro assay using labeled pyruvate.
The E1β subunit of pPDH is encoded by at least two genes in Arabidopsis designated E1β-1 and EE1β-2, which are about 95% identical at the amino acid level. The cDNA encoding the E1β-1 subunit of pPDH from Arabidopsis encodes a 44 kDa protein. The E1β-1 subunit of pPDH of Arabidopsis is 78% similar and 70% identical to the pPDH of Porphyra purpurea and 52% similar and 41% identical to the E1β subunit of mtPDH.
Also in this regard, the present invention further provides an isolated or purified nucleic acid molecule encoding the E3 subunit of a plant pPDH (E3 pPDH), such as that which is isolated from Arabidopsis, and a continuous fragment thereof comprising at least about 20 nucleotides. Preferably, the E3 pPDH-encoding nucleic acid molecule is (i) DNA and comprises SEQ ID NO: 27 (E3-1 pPDH) or SEQ ID NO: 29 (E3-2 pPDH) or a sequence that encodes SEQ ID NO: 28 (E3-1 pPDH) or SEQ ID NO: 30 (E3-2 pPDH), (ii) RNA and comprises a sequence encoded by SEQ ID NO: 27 or SEQ ID NO: 29 or a sequence that encodes SEQ ID NO: 28 or SEQ ID NO: 30, or (iii) a nucleic acid molecule that hybridizes to either one of the foregoing under stringent conditions. Also provided is an isolated or purified nucleic acid molecule encoding a modified E3 subunit of pPDH, which comprises one or more insertions, deletions and/or substitutions, and a continuous fragment thereof comprising at least about 20 nucleotides. Desirably, the modified E3 pPDH subunit does not differ functionally from the corresponding unmodified E3 pPDH subunit, such as that comprising SEQ ID NO: 28 or SEQ ID NO: 30. Preferably, the modified E3 subunit of a plant pPDH, together with the remaining unmodified subunits, does not differ functionally from a corresponding unmodified pPDH. Preferably, the modified pPDH converts pyruvate to acetyl-CoA at least about 50%, more preferably at least about 75%, most preferably at least about 90% as well as the corresponding unmodified pPDH, such as that comprising the E3 subunit comprising SEQ ID NO: 28 or SEQ ID NO: 30, as determined by in vitro assay using labeled pyruvate.
Yet another isolated or purified nucleic acid molecule encodes the A subunit of a plant ACL (ACL-A), such as that which is isolated from Arabidopsis, and a continuous fragment thereof comprising at least about 20 nucleotides. Preferably, the ACL-A-encoding nucleic acid molecule is (i) DNA and comprises SEQ ID NO: 7 or a sequence that encodes SEQ ID NO: 8, (ii) RNA and comprises a sequence encoded by SEQ ID NO: 7 or a sequence that encodes SEQ ID NO: 8, or (iii) a nucleic acid molecule that hybridizes to either one of the foregoing under stringent conditions. Also provided is an isolated or purified nucleic acid molecule encoding a modified A subunit of a plant ACL, which comprises one or more insertions, deletions and/or substitutions, and a continuous fragment thereof comprising at least about 20 nucleotides. Desirably, the modified ACL-A subunit does not differ functionally from the corresponding unmodified ACL-A subunit, such as that comprising SEQ ID NO: 8. Preferably, the modified ACL converts citrate to acetyl-CoA at least about 50%, more preferably at least about 75%, most preferably at least about 90% as well as the corresponding unmodified ACL, such as that comprising the A subunit comprising SEQ ID NO: 8, as determined by in vitro assay using labeled citrate.
In this regard, the present invention further provides an isolated or purified nucleic acid molecule encoding the B subunit of a plant ACL (ACL-B), such as that which is isolated from Arabidopsis, and a continuous fragment thereof comprising at least about 20 nucleotides. Preferably, the ACL-B-encoding nucleic acid molecule is (i) DNA and comprises SEQ ID NO: 9 (ACL-B1) or SEQ ID NO: 11 (ACL-B2) or a sequence that encodes SEQ ID NO: 10 or SEQ ID NO: 12, (ii) RNA and comprises a sequence encoded by SEQ ID NO: 9 or SEQ ID NO: 11 or a sequence that encodes SEQ ID NO: 10 or SEQ ID NO: 12, or (iii) a nucleic acid molecule that hybridizes to either one of the foregoing under stringent conditions. Also provided is an isolated or purified nucleic acid molecule encoding a modified B subunit of a plant ACL, which comprises one or more insertions, deletions and/or substitutions, and a continuous fragment thereof comprising at least about 20 nucleotides. Desirably, the modified ACL-B subunit does not differ functionally from the corresponding unmodified ACL-B subunit, such as that comprising SEQ ID NO: 10 or SEQ ID NO: 12. Preferably, the modified ACL converts citrate to acetyl-CoA at least about 50%, more preferably at least about 75%, most preferably at least about 90% as well as the corresponding unmodified ACL, such as that comprising the B subunit comprising SEQ ID NO: 10 or SEQ ID NO: 12, as determined by in vitro assay using labeled citrate.
ACL is encoded by a small gene family in Arabidopsis. The cDNA encoding ACL-A from Arabidopsis encodes a 45 kDa protein, whereas the cDNA encoding ACL-B from Arabidopsis encodes a 70 kDa protein. ACL-A is encoded by at least two genes in Arabidopsis which are designated ACL-A1 and ACL-A2. ACL-B is encoded by at least two genes in Arabidopsis which are designated ACL-B1 and ACL-B2. The ACL of Arabidopsis is 50% similar to that of the human and the rat.
An isolated and purified nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 15 or SEQ ID NO: 17 or encoding the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 18 is also provided by the present invention.
Likewise, an isolated and purified nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 21 or encoding the amino acid sequence of SEQ ID NO: 22 or a continuous fragment of either of the foregoing comprising at least about 20 nucleotides or a nucleic acid molecule that hybridizes to any of the foregoing under stringent conditions.
Yet another isolated or purified nucleic acid molecule provided by the present invention is that which encodes a plant ACH. Such a nucleic acid molecule, including a continuous fragment thereof of at least about 20 nucleotides in length, can be isolated from a plant in accordance with Example 9.
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 enzyme has enzymatic activity characteristic of the unmodified enzyme. 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 ACS, pPDH and ACL 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 molecules, it is preferred that the one or more substitution(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 molecules, a “continuous fragment of at least about 20 nucleotides of the isolated or purified nucleic acid molecule,” a given nucleic acid molecule is a continuous fragment that, for example, encodes an amino acid molecule that can carry out the same function as the corresponding complete amino acid molecule. For example, a fragment of an isolated or purified nucleic acid molecule encoding a plant ACS can be a continuous fragment of the ACS-encoding nucleic acid molecule that encodes an amino acid molecule that can convert acetate to acetyl-CoA in the presence of ATP and CoASH, but not necessarily as well as the corresponding complete amino acid molecule.
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, Wis., 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, as well as the additional nucleic acid molecules described below with respect to the present inventive method, 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); Swinbume 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 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 them 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 ColE1, 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, Calif.), the pET series (Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clonetech, Palo Alto, Calif.). Bacteriophage vectors, such as λGT10, λGT11, λZapII (Stratagene), λ EMBL4, and λ NM1149, also can be used. Examples of plant expression vectors include pBI101, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clonetech, Palo Alto, Calif.). Examples of animal expression vectors include pEUK-C 1, pMAM and pMAMneo (Clonetech).
A plant expression vector can comprise a native or nonnative promoter operably linked to a nucleic acid molecule encoding ACS, pPDH or ACL as described above. The selection of promoters, e.g., strong, weak, inducible, tissue-specific and developmental-specific, is within the skill in the art. Similarly, the combining of a nucleic acid molecule as described above with a promoter is also within the skill in the art.
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.
The present invention not only provides a vector comprising a nucleic acid molecule as described above but also provides a vector comprising or encoding an antisense sequence that hybridizes to or a ribozyme that cleaves an RNA molecule encoding a plant plastidic acetyl CoA synthetase, the E1α subunit of a plant plastidic pyruvate dehydrogenase, the E1β subunit of a plant plastidic dehydrogenase, the E2 subunit of a plant plastidic pyruvate dehydrogenase, the E3 subunit of a plant plastidic pyruvate dehydrogenase, the A subunit of a plant ATP-citrate lyase, the B subunit of a plant ATP-citrate lyase, a plant pyruvate decarboxylase, a plant acetyl CoA hydrolase, a plant mitochondrial pyruvate dehydrogenase, and a plant aldehyde dehydrogenase. The present invention also provides the antisense molecules, which preferably are at least about 20 nucleotides in length, and the ribozymes, which preferably comprise at least about 20 continuous nucleotides complementary to the target sequence on each side of the active site of the ribozyme.
In view of the above, the present invention provides a host cell comprising a vector as described above. In addition, the present invention provides a polypeptide produced by 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 addition to the above, the present invention provides polyclonal antibodies to ACS, pPDH E1α and E1β and ACL A and B. Such polyclonal antibodies can be produced in accordance with the methods of Examples 2, 4 and 6 or other methods known in the art.
In yet another embodiment, the present invention provides a method of altering the level of an enzyme 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 nucleic acid molecule selected from the group consisting of (i) a gene encoding an enzyme or, if the enzyme is comprised of subunits, a subunit of an enzyme selected from the group consisting of plastidic acetyl CoA synthetase, plastidic pyruvate dehydrogenase, ATP-citrate lyase, pyruvate decarboxylase, acetyl CoA hydrolase, mitochondrial pyruvate dehydrogenase and aldehyde dehydrogenase, (ii) a nucleic acid molecule comprising or encoding an antisense molecule to an RNA molecule transcribed from a gene of (i), and (iii) a nucleic acid molecule comprising a ribozyme to an RNA molecule transcribed from a gene of (i). The vector comprising a nucleic acid molecule of (i) increases the level of an enzyme in the plant cell, plant tissue, plant organ or plant, whereas the vector comprising a nucleic acid molecule of (ii) or (iii) decreases the level of an enzyme in the plant cell, plant tissue, plant organ or plant. Preferably, the alteration of the enzyme results in an alteration of the level of acetyl 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 acetyl 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 or one of those described below. 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 an enzyme 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. However, to the extent that antisense or ribozyme sequences are employed in the present inventive method, it would be advantageous to use a nucleic acid molecule isolated from a plant that is of the same origin as the plant cell, plant tissue, plant organ or plant in which the level of an enzyme is to be altered.
An isolated or purified nucleic acid molecule encoding PDC for use in the present inventive method can be obtained in accordance with methods known in the art. A cDNA clone encoding PDC 1 from maize is available from GenBank as accession number X177555 (genomic DNA X 59546). Partial clones Z21721 and L11312 for PDC-22 and partial clones Z21722 and L11313 for PDC-3 from maize are also available from GenBank. cDNA and deduced amino acid sequences for PDC-1 and PDC-2 from Arabidopsis are provided herein as SEQ ID NOS: 15-18 as set forth in FIGS. 8A-9B. Alternatively, an isolated or purified nucleic acid molecule encoding a PDC that has been modified by one or more insertions, deletions and/or substitutions, wherein the encoded PDC does not differ functionally from the unmodified PDC, can be used. Preferably, the modified PDC converts pyruvate to acetaldehyde preferably at least about 50%, more preferably at least about 75%, most preferably at least about 90% as well as the unmodified PDC as determined by in vitro assay using labeled pyruvate.
An isolated or purified nucleic acid molecule encoding the E2 subunit of pPDH for use in the present inventive method can be obtained in accordance with methods known in the art. A cDNA clone encoding E2 pPDH is available from Mooney et al., Biochemistry Department, University of Missouri (Mooney et al., Plant Physiol. 120: 443-451 (1999)). Alternatively, an isolated or purified nucleic acid molecule encoding the E2 subunit of pPDH that has been modified by one or more insertions, deletions and/or substitutions, wherein the encoded E2 subunit of pPDH does not differ functionally from the unmodified E2 subunit of pPDH, can be used. Preferably, a pPDH comprising a modified E2 subunit of pPDH converts pyruvate to acetyl-CoA preferably at least about 50%, more preferably at least about 75%, most preferably at least about 90% as well as a pPDH comprising an unmodified E2 subunit as determined by in vitro assay in the presence of labeled pyruvate.
An isolated or purified nucleic acid molecule encoding ACH can be isolated from a plant for use in the present inventive method using the ACH gene from Saccharomyces cervisiae (GenBank Accession No. M31036; Lee et al., J. Biol. Chem. 265: 7413-7418 (1990)). For example, existing Saccharomyces cerevisiae mutants (Lee et al., Biochim. Biophys. Acta 1297(1): 105-109 (1996); and Minet et al., Plant J. 2: 417-422 (1992)) can be complemented with a library expressing Arabidopsis cDNAs in a plasmid that can replicate and express those genes in yeast (see, for example, Wang et al., Plant Molec. Biol. 31: 1093-1104 (1996)). A clone that can complement the yeast mutant and restore wild-type growth capability is selected. Alternatively, a clone can be obtained by purifying the ACH to homogeneity and obtaining partial N-terminal sequence analysis. This sequence analysis is then reverse translated into a DNA sequence. The DNA sequence is then used to screen a cDNA library from Arabidopsis to find the clone containing the cDNA for ACH.
An isolated or purified nucleic acid molecule encoding mtPDH for use in the present inventive method can be obtained in accordance with methods known in the art. The mtPDH comprises four subunits. A cDNA clone encoding the E1α subunit of mtPDH from Arabidopsis is described in Luethy et al., Gene 164(2): 251-254 (1995). The cDNA clone comprises 1435 bp in which there is a 1167 bp open reading frame encoding a 43.0 kD polypeptide of 389 amino acids (pI 7.1). The E1α subunit from Arabidopsis is 47-51% identical at the amino acid level to other eukaryotic sequences. A cDNA clone encoding the E1β subunit of mtPDH from Arabidopsis is described in Luethy et al., Biochim. Biophys. Acta 1187(1): 95-98 (1994). The cDNA clone comprises 1320 bp in which there is a 1089 bp open reading frame encoding a polypeptide of 363 amino acids with a predicted molecular mass of 39,190 Da and an isoelectric point of 4.9. A 29 amino acid presumptive mitochondrial targeting sequence is present at the amino terminus. A cDNA clone encoding the E2 subunit (dihydrolipoamide acetyltransferase) of mtPDH from Arabidopsis is described in Guan et al., J. Biol. Chem. 270(10): 5412-5417 (1995). The cDNA clone comprises 2.2 kb. Such sequences can be used in the present inventive method.
Alternatively, an isolated or purified nucleic acid molecule encoding an E1α, E1β or E2 subunit of mtPDH that differs from the E1α or E1β subunits of the mtPDH of Luethy et al. or the E2 subunit of the mtPDH of Guan et al., respectively, by one or more insertions, deletions and/or substitutions, wherein the modified E1α, E1β or E2 subunit, together with the other unmodified subunits of mtPDH of Leuthy et al. or Guan et al., does not differ functionally from an unmodified mtPDH. Preferably, the modified subunit, together with the other unmodified subunits, converts pyruvate to acetyl CoA at least about 70%, preferably at least about 75%, more preferably at least about 85%, most preferably at least about 90% as well as the unmodified mtPDH comprising the subunits disclosed by Leuthy et al. and Guan et al. as determined by in vitro assay in the presence of labeled pyruvate can be used.
An isolated or purified nucleic acid molecule encoding ALDH for use in the present inventive method can be obtained in accordance with the methods set forth in U.S. Pat. No. 5,684,242 (the '242 patent). The '242 patent discloses the nucleic acid sequence of ALDH as SEQ ID NO: 1 and the corresponding amino acid sequence as SEQ ID NO: 2. cDNA and deduced amino acid sequences for ALDH-1, ALDH-2, ALDH-3 and ALDH-4 from Arabidopsis are provided herein as SEQ ID NOS: 19-26 in FIGS. 10A-13. Alternatively, an isolated or purified nucleic acid molecule encoding ALDH that differs from the ALDH of the '242 patent or the Arabidopsis ALDH sequences as set forth herein by one or more insertions, deletions and/or substitutions, wherein the encoded ALDH does not differ functionally from the preceding ALDH, can be used. Preferably, the modified ALDH converts acetaldehyde to acetate preferably at least about 50%, more preferably at least about 75%, most preferably at least about 90% as well as the unmodified ALDH as determined by in vitro assay in the presence of labeled acetaldehyde.
Preferred vectors for use in the present inventive method are characterized as described above. Like vectors, which comprise a nucleic acid encoding PDC, E2 pPDH, ACH, ALDH or mtPDH as described above, also are preferred. 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)).
If it is desired to increase the expression of a given gene, it is preferred to do so by introducing a gene encoding an enzyme or, if the enzyme is comprised of subunits, a subunit of an enzyme selected from the group consisting of plastidic acetyl CoA synthetase, plastidic pyruvate dehydrogenase, ATP-citrate lyase, aldehyde dehydrogenase, mitochondrial pyruvate dehydrogenase, pyruvate decarboxylase and acetyl CoA hydrolase. 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 the CaMV 35S promoter, 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. Desirably, expression of an enzyme comprising subunits is increased by increasing the expression of all subunits. Expression of an enzyme comprising subunits can be decreased by decreasing the expression of a single subunit.
In this regard, if expression is desired in a given tissue, a tissue-specific promoter can be used in the vector. Examples of tissue-specific promoters and enhancers include those described in Guerineau, Methods Mol. Biol. 49: 1-32 (1995); Meisel et al., Gent. Eng. 19: 183-199 (1997); and Edwards et al., Ann. Rev. Genet. 24: 275-303 (1990)). Similarly, an organ-specific promoter can be used in the vector. Developmentally specific promoters and inducible promoters also can be used, such as those described in Gruner et al., Eur. J Biochem. 220(1): 247-255 (1994); Caddick et al, Nat. Biotech. 16(2): 177-180 (1998); Moore et al., PNAS USA 95(1): 376-381 (1998); and Mett et al., PNAS USA 90(10): 4567-4571 (1993). Malate synthase and isocitrate lyase plant promoters are examples of developmentally specific promoters. Napin, phaseolin, oleosin, glycinin, cruciferin and betaconglycinin are examples of seed storage protein promoters. Inducible promoters, which can be used in those instances where the expression of a given gene is desired after a host plant has reached maturity, include temperature sensitive regulatory elements, heat shock promoters, stress response promoters, and chemically inducible promoters.
If it is desired to decrease the expression of a given gene, it is preferred to do so by introducing either a nucleic acid molecule comprising (i.e., in the case of an RNA vector) or encoding (i.e., in the case of a DNA vector) an antisense nucleic acid molecule to an RNA molecule transcribed from an aforementioned gene or a nucleic acid molecule comprising a ribozyme to an RNA molecule transcribed from such a gene (see, for example, Senior, Biotech. Genet. Eng. Rev. 15: 79-119 (1998); Bird et al., Biotech. Genet. Eng. Rev. 9: 207-227(1991); Matzke et al., Trends Genet. 11(1): 1-3 (1995); Baulcombe, Plant Mol. Biol. 32(1-2): 79-88 (1996); Castanatto et al., Crit. Rev. Eukaryot. Gene Exp. 2(4): 331-357 (1992); and Rossi, Trends Biotechnol. 13(8): 301-306 (1995)). In antisense technology, a nucleic acid segment from the desired plant gene can be cloned and operably linked to the promoter sequence such that the anti-sense strand of RNA is transcribed. For example, the 35S promoter from CaMV can be fused to a cDNA encoding a given enzyme but in opposite orientation from normal.
The nucleic acid sequence introduced in antisense suppression generally is substantially identical to at least a portion, preferably at least about 20 continuous nucleotides, of the endogenous gene or gene to be repressed, but need not be identical. The vectors can, thus, be designed such that the inhibitory effect applies to other proteins within a family of genes exhibiting homology or substantial homology to the target gene. The introduced sequence also need not be full-length relative to either the primary transcription product or fully processed mRNA. Generally, higher homology can be used to compensate for the use of a shorter sequence. Furthermore, the introduced sequence need not have the same intron or exon pattern, and homology of non-coding segments will be equally effective.
The plant cell, plant tissue, plant organ or plant is then contacted with the construct and the anti-sense strand of RNA is produced. In plant cells, it has been shown that anti-sense RNA inhibits gene expression (see, e.g., Sheehy et al., PNAS USA 85: 8805-8809 (1988); and Hiatt et al., U.S. Pat. No. 4,801,340). The resulting recombinant gene can be transformed into Arabidopsis plants, for example, using Agrobacterium-mediated transformation. Inhibition of expression of a given gene can be confirmed in a transformed plant cell by standard methods for measuring the presence and/or activity of a given enzyme. In this regard, it is important to point out that some plants, such as Arabidopsis, contain two genes, i.e., “paralogs,” encoding a given enzyme, such as ACL, PDC and ALDH. In such instances, it is desirable to decrease the expression of a given gene with antisense RNA because the paralogous genes generate mRNAs that are nearly identical in sequence (as is the case of ACL, PDC and ALDH mRNAs) and, therefore, a single antisense RNA molecule can reduce and even block the expression of both paralogs, depending on the antisense molecule utilized.
Ribozymes also have been reported to have use as a means to inhibit expression of endogenous plant genes. It is possible to design ribozymes that specifically pair with virtually any target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA. In carrying out this cleavage, the ribozyme is not itself altered and is, thus, capable of recycling and cleaving other molecules, making it a true enzyme. The inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the constructs. The design and use of target RNA-specific ribozymes is described in Haseloff et al., Nature 334: 585-591 (1988). Preferably, the ribozyme comprises at least about 20 continuous nucleotides complementary to the target sequence on each side of the active site of the ribozyme.
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.
Another method of decreasing acetyl CoA levels is co-supression. See, for example, Que et al., Dev. Genet. 22(1): 100-109 (1998) and Smyth, Curr. Biol. 7(12): R793-R795 (1997).
Alternatively, reverse genetics systems, which are well-known in the art, can be used to generate and isolate down-regulated or null mutants. One such system, the Trait Utility System for Corn, i.e., TUSC, is based on successful systems from other organisms (Ballinger et al., PNAS USA 86: 9402-9406 (1989); Kaiser et al., PNAS USA 87: 1686-1690 (1990); and Rushforth et al., Mol. Cell. Biol. 13: 902-910 (1993)). The central feature of the system is to identify Mu transposon insertions within a DNA sequence of interest in anticipation that at least some of these insertion alleles will be mutants. To develop the system in corn, DNA was collected from a large population of Mutator transposon stocks that were then self-pollinated to produce F2 seed. To find Mu transposon insertions within a specified DNA sequence, the collection of DNA samples is screened via PCR using a gene-specific primer and a primer that anneals to the inverted repeats of Mu transposons. A PCR product is expected only when the template DNA comes from a plant that contains a Mu transposon insertion within the target gene. Once such a DNA sample is identified, F2 seed from the corresponding plant is screened for a transposon insertion allele. Transposon insertion mutations of the an1 gene have been obtained via the TUSC procedure (Bensen et al., Plant Cell 7: 75-84 (1995)). This system is applicable to other plant species, at times modified as necessary in accordance with knowledge and skill in the art.
T-DNA insertional mutagenesis can be used to generate insertional mutations in one of the above-mentioned genes so as to affect adversely the expression of a given gene. Theoretically, about 100,000 independent T-DNA insertions are required for a 95% probability of getting an insertion in any given gene (McKinnet, Plant J. 8(4): 613-622 (1995); and Forsthoefel et al., Aust. J. Plant Physiol. 19: 353-366 (1992)). Currently, there are 12,000 such T-DNA-tagged lines that are publicly available. Additional T-DNA-tagged lines are being generated and are being made available. In this regard, Monsanto (St. Louis, Mo.) currently has a collection of T-DNA tagged lines, which are reported to contain 90,000-150,000 T-DNA insertions. T-DNA tagged lines of plants can be screened using PCR. For example, a primer can be designed for one end of the T-DNA and another primer can be designed for the gene of interest and both primers can be used in PCR. If no PCR product is obtained, then there is no insertion in the gene of interest. In contrast, if a PCR product is obtained, then there is an insertion in the gene of interest. Insertional mutations, however, often generate null alleles, which can be lethal. Alternatively, if there is more than one gene that encodes for a given enzyme, a mutation in one of the genes may not result in decreased expression of the enzyme encoded by the gene.
Another alternative method to decrease expression of a given gene is to use a compound that inhibits expression of one of the above-mentioned genes or that inhibits the activity of the enzyme encoded by one of the above-mentioned genes. For example, glucose is known to inhibit PDC.
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, deletions, duplications or replacements) 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 given gene involved in acetyl 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 a given enzyme can be measured by using labeled substrates in vitro. ACS activity can be assayed quickly and conveniently in vitro by incubating whole chloroplasts, chloroplast extracts or leaf extracts with labeled acetate, CoA, ATP and Mg and transferring aliquots of the reaction mixture to pieces of either Whatman No. 1 or DE81 filter paper as described by Roughan et al., Analyt. Biochem. 216: 77-82 (1994). The paper pieces are then washed to remove unreacted acetate. Acetyl CoA, which binds quantitatively to the paper, is then determined by scintillation counting. If desired, ACS can be further purified as described by Zeiher et al., Plant. Physiol. 96: 382-389 (1991) for use in in vitro assays.
Reducing pPDH in accordance with the above method is expected to result in a decrease in the pool of plastidic acetyl CoA, which, in turn, is expected to affect de novo fatty acid biosynthesis, i.e., synthesis of 18-carbon fatty acids. This would be most readily observable as a reduction in the accumulation of seed oils.
Reducing cytosolic ACL is expected to result in a decrease in the pool of cytosolic acetyl CoA, which, in turn, is expected to affect the biosynthesis of very long chain fatty acids and flavonoids. This would be most readily observable as a reduction of seed coat color (due to reduced flavonoids), a reduction of cuticle deposition on the aerial portion of the plant, and a reduction in 20- and 22-carbon fatty acids in the seed oil (due to reduced elongation of 18-carbon fatty acids).
Reducing PDC, ALDH and/or ACS is expected to affect the acetyl CoA pool. The reduction of ALDH is known to affect pollen development in plants (see U.S. Pat. No. 5,684,242).
Whether or not a given acetyl CoA-generating pathway affects another acetyl CoA pathway can be determined by measuring the effect on expression of acetyl CoA-generating genes and by measuring the generation of acetyl CoA pools in vivo. For example, using a transgenic plant, it can be determined whether or not the reduction in a given acetyl CoA generating enzyme causes compensatory alterations in the expression of other genes. As microarray chip technology becomes established and available (DeRisi et al., Science 278: 680-686 (1997)), the effect of a given genetic alteration on the expression of all cloned genes of Arabidopsis can be determined. Metabolic radiotracer studies can be performed to measure the generation of different acetyl CoA pools in vivo. In such studies, radioactively labeled precursors are provided to intact tissues and the radioactive label is monitored as the precursor is metabolized. By comparing wild-type plants and plants that have reduced activities of one of the acetyl CoA-generating enzymes, the source of different acetyl CoA pools can be determined as well as the effect of the reduction in a given acetyl CoA generating enzyme.
In addition to being useful in the study of acetyl CoA generation in plants, including the spatial and temporal patterns of expression of genes encoding enzymes required for acetyl CoA generation, the above-described method is useful in the generation of plants for the production of acetyl CoA-derived phytochemicals. The method is useful in the alteration of acetyl 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 the acetyl CoA level in a given plant (or plant cell, tissue or organ) is different as a result of the practice of the present inventive method as compared to a like plant, the acetyl CoA level of which has not been altered as a result of the practice of the present inventive method.
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 an enzyme 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 an enzyme has been altered. Preferably, the alteration of the level of an enzyme results in an alteration of the level of acetyl CoA.
The above method can be adapted for in vitro production of acetyl CoA, which, in turn, can be used to produce acetyl CoA phytochemicals. For example, the various enzymes required for acetyl 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 acetyl 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 cloning of ACS cDNA from Arabidopsis and the comparison of its sequence with ACS from E. coli and S. cerevisiae. 
The deduced amino acid sequences of the ACS genes from E. coli and S. cervisiae were used to search the dbEST database (National Library of Medicine, National Institutes of Health, Bethesda, Md.) using the program BLAST. One Arabidopsis thaliana cDNA clone was identified as a possible ACS gene. This clone, 220J9T7 (accession number N38599), was obtained from the Arabidopsis Biological Research Center (ABRC) at Ohio State University.
Plasmid DNA from this clone was prepared and sequenced by the Biotechnology Instrumentation Facility of Iowa State University. Analysis of the sequence data revealed that this clone (J9) encoded approximately 40% of the C-terminal portion of ACS. Repeated searches of the dbEST database failed to reveal any longer clonal sequences.
cRACE (Maruyama et al., Nucleic Acids Research 23: 3796-3797 (1995)) was used to isolate the rest of the ACS gene. mRNA from Arabidopsis thaliana was prepared from fresh plant tissue. Nesting ACS-specific primers, based on the 5′ end sequence of J9, were used in two sequential cDNA synthesis reactions to generate a specific single-stranded, antisense cDNA fragment encoding a fragment of ACS DNA 5′ to the known J9 sequence. Ligation and circularization of this single-stranded cDNA by RNA ligase was followed by two sequential PCR amplifications, using nesting primer pairs based on J9 sequence. This process yielded an approximately 320 bp fragment of DNA encoding a further portion of the ACS gene. This fragment was cloned into the vector pBS (Stratagene), named J9E1, and sequenced.
The remainder of the ACS gene was obtained by PCR amplification of an Arabidopsis thaliana cDNA library constructed in the vector pSPORT (Life Technologies, Grand Island, N.Y.). Two nesting antisense primers were designed and synthesized that hybridize to the 5′ terminus of J9E1. These primers were used in conjunction with two nesting sense vector primers specific to the vector pSPORT to amplify sequentially by PCR a further 270 bp fragment of ACS cDNA. This fragment was cloned in pBS, given the name J9E2, and sequenced.
Likewise, two more nesting antisense primers were designed and synthesized that corresponded to the 5′ terminus of J9E2. Sequential PCR amplification of the cDNA library with these primers and the vector primers yielded a cDNA fragment encoding the rest of the ACS gene. This fragment was cloned in pBS, named J9E3, and sequenced.
All of the sequence data from clones J9, J9E1, J9E2 and J9E3 were aligned and condensed to give a single DNA sequence encoding the complete Arabidopsis thaliana ACS gene. This sequence was submitted to GenBank as accession no. AF036618. This DNA encodes a protein of 693 amino acids and has a calculated molecular weight of 76,678 daltons.
MACAW alignment of the cDNA sequence of ACS from Arabidopsis with the protein sequence of ACS from E. coli and the protein sequences of the two isoforms of ACS from S. cerevisiae shows that the ACS from Arabidopsis is 53 % identical to the ACS from E. coli and 37% identical to the I isoform of ACS from S. cerevisiae and 40% identical to the II isoform of ACS from S. cerevisiae. 
Example 2
This example describes the production of a polyclonal antibody to ACS from Arabidopsis.
PCR primers were designed such that the coding region of clone J9 would be amplified by PCR as a DNA fragment terminated by restriction sites suitable for cloning into the E. coli protein expression vector pMALC-2 (New England Biolabs, Beverly, Mass.). The expression vector clone J9-NT1/pMALC-2 was constructed according to the instructions furnished by the supplier of the vector. This clone, when transformed into E. coli and induced with isopropylthio-β-galactoside (IPTG), caused the production of a recombinant chimeric fusion protein consisting of the C-terminal portion of Arabidopsis thaliana ACS fused to the E. coli maltose binding protein (MBP). This recombinant protein was purified from one liter cultures of transformed E. coli and purified using the amylose column supplied by New England Biolabs, according to the manufacturer's protocols. The purified protein was assayed electrophoretically for purity, quantitated, and given to the Biotechnology Instrumentation Facility of Iowa State University for production of polyclonal antibodies in rabbits. Pre-immune and immune sera from the rabbits were tested for specificity and titer by immunoblotting against the original antigen recombinant protein.
Example 3
This example describes the cloning of cDNA for the E1α, E1β and E3 subunits of pPDH and the comparison of the sequence of the cDNA encoding the E1β subunit with the sequence of pPDH from red algae and mtPDH from Arabidopsis. 
The dbEST database was searched with the deduced amino acid sequences of the E1α and E1β subunits of PDH from Porphyra purpurea using the program BLAST. Clones identified as possible pPDHs were obtained from the ABRC at Ohio State University.
Arabidopsis thaliana EST clone 232D14T7 (N65567) and 232D13T7 (N65566) displayed a significant degree of homology to the E1α subunit of PDH from Porphyra purpurea. Both clones contain identical nucleotide sequences encoding all but the first 50-70 amino acids of the E1α subunit of pPDH, based on homology to other E1α subunits of PDH. Clone 232D14T7 (D14) was chosen for sequencing. The DNA sequence of this clone comprises SEQ ID NO: 3 and the deduced amino acid sequence comprises SEQ ID NO: 4 (see FIG. 2 and Sequence Listing).
Arabidopsis thaliana EST clones 163C12T7 (R2996; E1β-1) and 169K3T7 (R64987; E1β-1) displayed a significant degree of homology to the E1β subunit of PDH of Porphyra purpurea. These two clones are related to, but not identical to, the corresponding mtPDH of Arabidopsis thaliana. Clone 163C12T7 (C12) was chosen for sequencing. Clone C12 appears to encode almost all of the coding region of the E1β subunit of pPDH. The sequence of this clone comprises SEQ ID NO: 5 and the deduced amino acid sequence comprises SEQ ID NO: 6 (see FIG. 3 and Sequence Listing). A second E1β subunit gene (E1β-2) was discovered in Arabidopsis by searching through the Arabidopsis genome sequence in Genbank. Careful analysis showed that the genomic sequence in the database was different from the cDNA sequence of E1β-1. Thus, all of the cDNA EST clones were requested from the Arabidopsis Biological Resource Stock Center (Ohio State University, Columbus, Ohio) and sequenced. The cDNA sequences of E1β-1 and E1β-2 are nearly identical.
Genes encoding the E3 subunit of pPDH from Arabidopsis were isolated as follows. The Arabidopsis EST database was searched using the E3 gene for the cyanobacterium Synechocystis PCC 6803 (Engels et al., Microbiology 143: 3543-3553 (1997)). Two partial cDNA clones were obtained from the Arabidopsis Biological Resource Stock Center. The sequences were used as probes to screen a cDNA library. Near full-length clones were isolated. The genes were translated and transcribed in vitro and to verify that the genes were plastidic as opposed to mitochondrial. The translated proteins were imported into isolated chloroplasts and processed to mature form.
MACAW alignment of the cDNA sequence of E1β pPDH from Arabidopsis with the protein sequence of pPDH from red algae and the protein sequence of the mtPDH from Arabidopsis shows that the pPDH from Arabidopsis is 68% identical to the pPDH from red algae and 37% identical to the mtPDH from Arabidopsis. 
Example 4
This example describes the production of polyclonal antibodies to the E1α and E1β subunits of chloroplastic PDH.
Primers were designed such that the coding regions of D14 and C12 of Example 3 were incorporated into the E. coli expression vector pET24-a. After assembly of these two expression constructs, an expression cassette consisting of the vector promoter, the C12 coding sequence, and the vector terminator was excised from the C12 expression plasmid and incorporated into the D14 expression plasmid, yielding a tandem expression plasmid capable of simultaneously expressing both subunits of pPDH. These recombinant pPDHs were expressed in E. coli as insoluble inclusion bodies. These inclusion bodies were purified to near homogeneity. The resulting protein was sent to the Biotechnology Instrumentation Facility of Iowa State University for production of polyclonal antibodies in rabbits. Pre-immune and immune sera from the rabbits were tested for specificity and titer by immunoblotting against the original antigen recombinant proteins.
Example 5
This example describes the cloning of cDNA for the A and B subunits of ACL from Arabidopsis. 
The cDNA clones pACL-A1 and pACL-B1 were identified by sequence similarity searches of the Arabidopsis EST (Expressed Sequence Tags) database. The EST cDNA pACL-A1 (clone ID TASG097, Genbank accession #Z18045) was identified because the sequence of its 5′-end was similar to the sequence of the 5′-end of the human ATP-citrate lyase. The EST cDNA pACL-B1 (clone ID VBVYC01, Genbank accession #Z33810) was identified because the sequence of its 5′-end was similar to the sequence near the middle of the human ATP-citrate lyase. These two clones were obtained from the Arabidopsis Biological Resource Center (Ohio State University). Both strands of both clones were sequenced at the Iowa State University DNA Sequencing Facility, using an ABI automatic sequencer.
The ACL-A2 sequence was identified by a BLAST sequence search of the publicly available data generated by the Arabidopsis Genome Initiative (http://genome-www.stanford.edu/Arabidopsis/agi.html). ACL-A2 is an Arabidopsis gene that is paralogous to the ACL-A1 cDNA. That is, the ACL-A2 gene codes for the same protein as the ACL-A1 cDNA, but based on the fact that the 5′- and 3′-untranslated regions of these two sequences are different, these two sequences represent different members of the ATP-citrate lyase gene family.
The ACL-B2 sequence was identified by screening an Arabidopsis cDNA library prepared from polyA RNA isolated from developing siliques of Arabidopsis (Castle and Meinke, Oklahoma State University, Stillwater) in the vector lambda gt10 with radioactively labeled ACL-B1 cDNA as described by Sambrook et al. (1989), supra. Briefly, approximately 200,000 recombinant phage from the library were grown on petri plates and replicated to nitrocelluose membranes. Replica filters were incubated at 65° C. in hybridization solution (5×SSC, 1×Denhardt's solution, 0.2% (w/v) SDS, 10 mM EDTA, 0.1 mg/ml salmon sperm DNA, 10% (w/v) dextran sulfate, 50 mM Tris-HCl, pH 8.0) with 32P-labeled probe for 12 hr. After hybridization, filters were washed at 65° C. in 2×SSC, 0.5% (w/v) SDS, and subsequently with 0.1×SSC and 0.1% (w/v) SDS. Recombinant plaques that hybridized with the probe were plaque-purified and a cDNA insert was subcloned into the plasmid vector pBSK for sequencing purposes. The resulting sequence was termed ACL-B2, which is a full-length cDNA clone coding for the ACL-B subunit and is encoded by a separate gene from ACL-B 1. The nucleotide sequences of ACL-B 1 and ACL-B2 share about 80% identity and encode polypeptides that are over 95% identical.
ACL-A1, ACL-A2, ACL-B1 and ACL-B2 share a high degree of sequence identity with rat and human ACL. The human ACL mRNA is 4.3 kb in length and codes for a protein of 1100 residues. The ACL-A1 mRNA is 1.5 kb in length and codes for a protein of 423 residues that is 50% identical to the N-terminus of the human ACL. The ACL-A2 gene codes for a mRNA that is a paralog of the ACL-A1 mRNA; they each code for proteins that are over 98% identical even though their nucleotide sequences are only about 80% identical and their 5′- and 3′-untranslated regions are distinct. The ACL-A2 gene comprises 12 exons and 13 introns. The partial ACL-B 1 cDNA is 1.03 kb in length and codes for a polypeptide of 272 residues that is 50% identical to the C-terminus of the human ACL. The full-length ACL-B1 mRNA is 2.2 kb in length.
The deduced ACL-A and ACL-B polypeptides have no recognizable targeting sequence and, as with the rat and human ACL polypeptides, they are cytosolic.
Numerous sequence databases were searched for additional orthologs of the ACL-A and ACL-B polypeptides. No orthologs were identifiable in the entire genome of the yeast Saccharomyces cerevisiae, which is consistent with the absence of ACL activity in this species (Kohlhaw and Tan-Wilson, J. Bacteriol. 129: 1159-1161 (1977)). However, orthologs of ACL-A and ACL-B have been identified in the yeast Schizosaccharomyces pombe. The ACL-A ortholog is a clone of a gene that resides on chromosome 1 of S. pombe, and its amino acid sequence is over 60% identical to Arabidopsis ACL-A. The ACL-B ortholog from S. pombe is a partial cDNA clone, and its amino acid sequence shares 60% identity with that of the Arabidopsis ACL-B. Both S. pombe orthologs have been identified as ATP citrate lyases due to the high degree of sequence identity to the animal ACL.
The ACL-B and ACL-A mRNAs are transcribed from two different genes. Evidence for this conclusion comes from a series of experiments. First, Arabidopsis DNA was digested with Xba I, Sac I, Hin dIII, Eco RI, and Bam HI and subjected to Southern blot hybridization using as a probe the ACL-A1 cDNA and the ACL-B cDNA. Hybridization analyses of the Arabidopsis DNA probed individually with the two cDNAs revealed that each cDNA hybridized to a nonoverlapping set of restriction fragments. Second, screening of a lambda-based Arabidopsis genomic library with pACL-A and pACL-B resulted in the isolation of two nonoverlapping sets of genomic clones that hybridize with each of the two cDNAs. Restriction digests and Southern blot analyses of the 12 genomic clones that hybridize to pACL-A enabled subclassification of the clones into two groups, which is consistent with the data from the genomic Southern blot that indicates that the ACL-A mRNA is encoded by a small gene family. Based on similar analyses, the eight pACL-B hybridizing genomic clones were subclassified into three groups, which is consistent with the data from the genomic Southern blot that indicates that the ACL-B mRNA is encoded by a small gene family. Third, the sequence of a BAC clone containing one of the ACL-A genes (ACL-A1) indicates that an ACL-B gene is not within 10 kb of the ACL-A1 gene. The ACL-A1 gene is located on chromosome 1 of Arabidopsis, and the gene sequence is interrupted by 11 introns.
Example 6
This example describes the production of polyclonal antibodies to the A and B subunits of ACL.
The ACL-A and ACL-B sequences were expressed in E. coli using the pET 30 expression vector (Novagen, Madison, Wis.). The expressed proteins were then purified by preparative SDS-PAGE and were used to immunize rabbits to obtain antisera.
These antisera were used to probe Western blots of Arabidopsis seedling extracts. Anti-ACL-A detected a single polypeptide of 45-kDa, which is very close to the molecular weight predicted from the sequence of pACL-A (which encodes for a 423 residue polypeptide). Anti-ACL-B antibodies detected a polypeptide of about 70-kDa, which is close to that predicted based upon the size of the ACL-B mRNA (2.2-kb).
Example 7
This example describes how the cDNA encoding ACH from Saccharomyces cerevisiae can be used to isolate the mtACH gene from a plant.
A cDNA clone encoding a plant mtACH can be identified by sequence similarity searches of the Arabidopsis EST (Expressed Sequence Tags) database (Arabidopsis Genome Initiative) to the yeast ACH cDNA. Positive clones can then be obtained from the ABRC (Ohio State Resource Center). Both strands of the positive clones can then be sequenced, for example by using an ABI automatic sequencer.
Given that recent efforts to identify a mtACH EST from this database have not yielded any positive clones (which could be due to the fact that the Arabidopsis Genome Initiative only contains about 20% of the genome), a plant mtACH gene can be cloned from Arabidopsis by complementation of the existing Saccharomyces cerevisiae mutants (Lee et al. (1990), supra). The mutant can be transformed with a library expressing Arabidopsis cDNAs in a plasmid that would replicate and express those genes in yeast (Minet et al., Plant J. 2: 417-422 (1992)). A clone can then be selected that complements the Saccharomyces mutant and restores (e.g., wild-type) growth capability. The Arabidopsis cDNA for ACH can then be copied by PCR and the DNA fragment can then be cloned in pBS for sequencing.
Alternatively, antisera to the yeast ACH can be generated and used to isolate mtACH from a plant. The amino acid sequence of the isolated mtACH can the be determined in whole or in part. The sequence can then be used to generate nucleic acid probes for screening a cDNA library or a genomic library in order to isolate the mtACH gene from plants. The mtACH gene from plants can then be sequenced.
If desired, antisera against the isolated plant mtACH can be used to screen an expression library for expression of mtACH. Antisera can be produced in rabbits using transgenically expressed protein from E. coli. For example, the Arabidopsis cDNA for ACH can be cloned into the pET24 expression vector as a translational fusion. The plasmid can then be transformed into E. coli, where it would express the protein. The protein, which accumulates as an inclusion body within the bacterium, can then be purified by centrifugation and SDS-gel electrophoresis. The protein can then be supplied to the Biotechnology Instrumentation Facility at Iowa State University for the production of polyclonal antibodies in rabbits. Pre-immune and immune sera from rabbits can then be tested for specificity and titer by immunoblotting against the original antigen recombinant protein.
Example 8
This example describes the cloning of ALDH cDNAs from Arabidopsis. 
The deduced amino acid sequence of the maize mitochondrial ALDH, coded by the rf2 gene (Cui et al., Science 272: 1334-1336 (1996)) was used to search the dbEST database (National Library of Medicine, National Institutes of Health, Bethesda, Md.) using the program BLAST. This resulted in the identification of 7 Arabidopsis EST cDNA clones (Genbank Accession #R83958, Z26417, N96630, T13678, R86795, AA041030, AA395226), which were obtained from the Arabidopsis Biological Research Center (ABRC) at Ohio State University, and they were each sequenced. All seven clones were partial cDNA. Using these EST cDNAs as probes, Arabidopsis cDNA libraries were screened as described in Example 5. This resulted in the isolation of full-length cDNA clones that corresponded to four gene products, ALDH1, ALDH2, ALDH3, ALDH4. The nucleotide and deduced amino acid sequences of the Arabidopsis ALDH1 (FIGS. 10A and 10B), ALDH2 (FIGS. 11A and 11B), ALDH3 (FIGS. 12A and 12B) and ALDH4 (FIG. 13) are presented. BLAST sequence searches of the publicly available data generated by the Arabidopsis Genome Initiative, with ALDH1, ALDH2, ALDH3 and ALDH4 nucleotide sequences confirmed that these cDNAs are products of four distinct genes. These genes occur on chromosomes 3, 3, 1 and 4, respectively, of Arabidopsis. Of these sequences, ALDH1 and ALDH3 encode the most similar proteins (they share 85% sequence similarity), whereas the similarity between ALDHI and ALDH3 is lower (66%), and the similarity between ALDH1 and ALDH4 is even lower (40%).
Example 9
This example describes the cloning of PDC cDNAs from Arabidopsis. 
The nucleotide sequences of the two Arabidopsis PDC genes (PDC1, Genbank accession #U71122, and PDC2, Genbank accession #U71122) were used to search the dbEST database (National Library of Medicine, National Institutes of Health, Bethesda, Md.) using the program BLAST. This resulted in the identification of 6 Arabidopsis EST cDNA clones, which were obtained from the Arabidopsis Biological Research Center (ABRC) at Ohio State University, and they were sequenced. All six clones were partial cDNA clones, three of which (Genbank accession #F14476, T04727 and F14475) matched the PDC1 gene sequence, and the other three (Genbank accession #N97215, Z35007 and AA597828) matched the PDC2 gene sequence. Using these EST cDNAs as probes, Arabidopsis cDNA libraries were screened as described in Example 5. This resulted in the isolation of full-length cDNA clones corresponding to the PDC1 and PDC2 genes. The nucleotide and deduced amino acid sequences of the Arabidopsis PDC1 (FIG. 8) and PDC2 (FIG. 9) are presented. The PDC1 and PDC2 cDNAs share 75% sequence identity, and they encode proteins that share 82% sequence identity or 87% sequence similarity. BLAST sequence searches of the publicly available data generated by the Arabidopsis Genome Initiative with PDC1 and PDC2 nucleotide sequences confirmed that these cDNAs are products of two distinct genes. These genes occur on chromosomes 4 and 5, respectively, of Arabidopsis. 
Example 10
This example describes the accumulation of ACL-A, ACL-B, PDH and ACS mRNAs determined by RNA blot analysis during stages of plant development.
RNA was extracted from Arabidopsis leaves, buds, flowers, and siliques as described previously (Weaver et al., Plant Physiol. 1 10: 1021 (1995)). Radioactive ACL-A and ACL-B RNAs were obtained by in vitro transcription from the respective pBSK clones. The RNA concentrations were determined from the absorbance at 280 and 260 nm.
Ten μg 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×SSC, 2% SDS for 10 min at room temperature, and then washed twice with 0.1×SSC, 0.1% SDS for 20 min at 65° C. The membranes were exposed to a phosphor screen (Molecular Dynamics, Sunnyvale, Calif.) for 4 hr, and the radioactivity in each band was quantified with a Storm 840 Phosphorlmager (Molecular Dynamics).
The accumulation patterns of the ACL-A and ACL-B mRNAs were identical at the level of resolution afforded by these techniques. During silique development, ACL-A and ACL-B mRNAs accumulated to the highest levels in flower buds and in developing siliques about 1-4 days after flowering. However, both ACL-A and ACL-B levels tapered off in the siliques at around 8 days after flowering and were barely detectable in the siliques at 15 days after flowering.
The absolute level of PDH E1β subunit mRNA accumulation (per total RNA) was greater than that of ACS mRNA in siliques at all stages of development. PDH E1β subunit mRNA accumulation was very high in siliques containing seeds undergoing rapid oil accumulation (5-7 days after flowering).
Example 11
This example describes the spatial and temporal patterns of expression of pPDH, ACS and ACL determined by in situ hybridization.
Arabidopsis siliques (1 to 13 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 ACL-A, ACL-B, E1β pPDH or ACS cDNA (cloned in pBluescript SK). The labeled probes were hybridized to the tissue sections by 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.
Using in situ hybridization to RNA, the spatial and temporal patterns of expression of pPDH, ACS, ACL-A and ACL-B were determined and were found to be heterogeneous. Accumulation of PDH mRNA was very high in torpedo-staged and walking-staged embryos, the stages in which oil accumulation is also highest. The pattern was almost identical to that of heteromeric acetyl CoA carboxylase (ACC). These results implicate pPDH as the source of the plastidic pool of acetyl CoA. In contrast to the pPDH E1β subunit mRNA, the mRNA coding for ACS accumulated to only very low levels in embryos of Arabidopsis, with maximal accumulation occurring in heart stage embryos, after which accumulation decreases. Instead, the ACS mRNA accumulated to the highest levels in the root tips of radicles of embryos in seeds from 1-4 days after imbibition, in anther filaments (especially near the junction of the filament and anther), and in the funiculus throughout seed development. Very high expression of ACS in filaments is consistent with some role of plastidic acetyl CoA in this organ. While ACS does not appear to be important in providing acetyl CoA for fatty acid synthesis for oils, the spatial and temporal expression of pPDH is consistent with this enzyme being associated with the production of acetyl CoA for oil biogenesis in developing seeds. The spatial and temporal patterns of ACL-A and ACL-B mRNAs were identical at the level of resolution afforded by these techniques. ACL-A and ACL-B mRNAs accumulated to high levels in the inner integuments of ovules the day preceding testal (seed coat) deposition; in epidermal cells of growing organs; in tapetal cells of anthers; and in epidermis and trichomes of young leaves. The co-accumulation of ACL-A and ACL-B with the cytosolic ACC indicates that ACL generates the cytosolic pool of acetyl-CoA. The higher level of accumulation of mRNAs in the epidermis may be associated with cuticular wax formation. The-co-accumulation of the ACL-A, ACL-B and ACC mRNAs in the inner integument a day preceding deposition of the testa may be associated with deposition of the flavonoid polymers phlobaphens (Stafford, “Metabolism and regulation of phenolics: gaps in our knowledge,” in Phytochemicals and Health, D. L. Gustine and H. E. Flores, editors. Amer. Soc. Plant Physiol. (1995)).
Example 12
This example describes the effect of immunoprecipitation of ACL-A and ACL-B polypeptides from Arabidopsis extracts on ACL activity.
The antibodies of Example 6 can be used to determine the effect of immunoprecipitation on ACL activity in extracts of Arabidopsis. Aliquots of Arabidopsis extracts will be mixed with increasing quantities of each antiserum or with control preimmune serum. Following an incubation on ice, antigen-antibody complexes will be bound to Protein A-Agarose beads, which will be pelleted by centrifugation. The supernatant solutions will be assayed for ACL activity.
ACL enzymatic activity can be determined by adaptation of a spectrophotometric assay that was initially developed for animals (Takeda et al., Meth. Enzymol. 27: 153-160 (1969)) and has since been used to characterize ACL in extracts of pea (Kaethner and ap Rees, Planta 163: 290-294 (1985)) and Brassica (Ratledge et al., Lipids 32: 7-12 (1997)). The assay couples the rate of appearance of oxaloacetate production to the oxidation of NADH, with the enzyme malate dehydrogenase, resulting in a measurable change in absorbance at 340 nm.
Example 13
This example describes how the level of acetyl CoA in a plant can be increased by increasing the copy number of one or more genes involved in acetyl CoA production.
The level of acetyl CoA generated in a cell of a plant can be increased by increasing the accumulation of one or more of the enzymes involved in the generation of acetyl CoA. This can be achieved by introducing additional copies of the one or more genes into the genome of the organism. A copy of each acetyl CoA-producing 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 acetyl CoA-producing gene. Alternatively, multiple copies of an acetyl CoA-producing gene are cloned into the transforming vector. Alternatively, an acetyl CoA-producing gene is cloned into transformation vectors that carry different selectable marker genes and multiple transformations are carried out to introduce multiple copies of an acetyl CoA-producing gene. In some cases, it is necessary to introduce a combination of acetyl CoA-producing genes. This is achieved by cloning a combination of acetyl CoA-producing genes into the same transformation vector or into different transformation vectors that carry different selectable marker genes.
Example 14
This example describes how the level of acetyl CoA in a plant can be increased by increasing the expression of one or more genes involved in acetyl CoA production.
The level of acetyl CoA generated in a cell of a plant can be increased by increasing the accumulation of one or more of the acetyl CoA-producing enzymes. This can be achieved by introducing into the genome of an organism copies of one or more acetyl CoA-producing genes or cDNAs fused to novel expression regulatory sequences that express the acetyl CoA-producing gene(s) at higher levels than normal. A copy of the acetyl CoA-producing 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 acetyl CoA-producing gene or combinations of novel acetyl CoA-producing genes can be introduced into the genome of an organism as described in Example 13.
Example 15
This example describes how the level of acetyl CoA in a plant can be decreased by using antisense technology.
The level of acetyl CoA generated in a cell of a plant can be decreased by decreasing the accumulation of one or more enzymes involved in acetyl CoA production. This can be achieved by introducing into the genome a transgene that expresses an antisense RNA of one or more enzymes involved in acetyl CoA production. The antisense RNA gene consists of a cDNA coding for an acetyl CoA producing enzyme, fused to upstream (5′) and/or downstream (3′) transcriptional or translational regulatory sequences with the cDNA being in the opposite orientation from the norm. This antisense gene is cloned into an appropriate transformation vector and transformed into a genome as described in Example 14. Multiple copies of the antisense gene can be introduced into a genome as described in Example 14. Antisense genes for a combination of acetyl CoA-generating enzymes can be introduced into a genome as described in Example 14.
Example 16
This example describes how to decrease the level of acetyl CoA in a plant using ribozymes.
The level of acetyl CoA generated in a cell of a plant can be decreased by decreasing the accumulation of one or more acetyl CoA-producing enzymes. This can be achieved by introducing into the genome a transgene that expresses a ribozyme targeted against a mRNA coding for an acetyl CoA-producing enzyme. The ribozyme-containing gene consists of the full-length or partial cDNA coding for an acetyl CoA-generating enzyme in opposite orientation from the norm into which a ribozyme sequence is inserted. This ribozyme containing cDNA is fused to upstream (5′) and/or downstream (3′) transcriptional or translational regulatory sequences. This ribozyme containing gene is cloned into an appropriate transformation vector and transformed into a genome as described in Example 14. Multiple copies of the ribozyme-containing gene can be introduced into a genome as described in Example 14. Ribozyme-containing genes targeted against a combination of acetyl-CoA-generating enzymes can be introduced into a genome as described in Example 14.
Example 17
This example describes how the level of acetyl CoA in a plant can be increased or decreased by using gene replacement.
The level of acetyl CoA generated in a cell of a plant can be altered by altering the activity of one or more acetyl CoA-producing enzymes. This can be achieved by a gene replacement method via homologous recombination. In this method, the endogenous acetyl CoA-producing gene is replaced by a mutagenized acetyl-CoA-producing gene. The mutagenized acetyl CoA-producing gene codes for an acetyl CoA-producing enzyme that is either more or less efficient in catalysis than the one encoded by the endogenous, replaced gene. The acetyl CoA-producing gene is mutagenized by one or more nucleotide deletions, insertions, duplications or replacements. 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 18
This example describes how to decrease the level of acetyl CoA in a plant by using co-suppression.
The level of acetyl CoA generated in a cell of a plant can be decreased by decreasing the accumulation of one or more of the acetyl CoA-producing enzymes. This can be achieved by co-suppression. For example, the cDNA coding for an acetyl-CoA-producing enzyme 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. 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 acetyl-CoA producing gene. To identify these co-suppressing plants, extracts from at least 100 transgenic plants will be analyzed for the enzymatic activity of the acetyl-CoA-producing enzyme.
Example 19
This example describes how to increase acetyl-CoA levels by overexpressing an acetyl-CoA-producing enzyme (eg., ACS) in a model organism (i.e., Arabidopsis).
The full-length ACS cDNA is cloned into a plant expression vector such as pB1101, down-stream of the Cauliflower Mosaic Virus 35S RNA promoter (CaMV, 35S promoter). 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 ACS cDNA and the CaMV, 35S promoter as probes.
Transgenic plants are tested for expression of the ACS transgene, for increased ACS activity and for increased accumulation of acetyl-CoA.
Expression of the ACS transgene is carried out by analyzing the accumulation of the ACS mRNA and polypeptide. The ACS mRNA can be detected by Northern hybridization with the ACS cDNA, or by RNase protection assays using an ACS transgene-specific probe.
The ACS polypeptide is detected by Western blot analysis of total proteins separated by SDS-PAGE and probed with ACS-specific antibodies. ACS activity is determined by incubating an extract with labeled acetate, in the presence of CoA and ATP, and monitoring the production of labeled acetyl-CoA. The accumulation of acetyl-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 acetyl-CoA is identified by co-elution with authentic acetyl-CoA. Acetyl-CoA concentration is determined based on absorbance at 254 nm.
Example 20
This example describes how to decrease acetyl-CoA levels by expressing an antisense RNA for an acetyl-CoA-producing enzyme (eg., ACS) in a model organism (i.e., Arabidopsis).
The full-length (or partial fragment) ACS cDNA is cloned into a plant expression vector such as pBI101, down-stream of the Cauliflower Mosaic Virus 35S RNA promoter (CaMV, 35S promoter), but in opposite orientation from normal. 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 minute 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 ACS cDNA and the CaMV, 35S promoter as the probe.
Transgenic plants are tested for expression of the antisense ACS transgene, for decreased accumulation of the ACS polypeptide, for decreased ACS activity, and for decreased accumulation of acetyl-CoA.
Expression of the antisense ACS transgene is carried by analyzing the accumulation of the ACS antisense RNA. RNA is extracted from transgenic seedlings and the ACS antisense RNA is detected by Northern hybridization, or by RNase protection assays using an ACS sense strand riboprobe.
The ACS polypeptide is detected by Western blot analysis of total proteins separated by SDS-PAGE and probed with ACS-specific antibodies. ACS activity is determined by incubating an extract with labeled acetate, in the presence of CoA and ATP, and monitoring the production of labeled acetyl-CoA.
The accumulation of acetyl-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 acetyl-CoA is identified by co-elution with authentic acetyl-CoA. Acetyl-CoA concentration is determined based on absorbance at 254 nm.
Example 21
This example describes the alteration of ACS levels in a plant using sense and antisense nucleic acids.
The full-length ACS cDNA was cloned into the plant expression vector designated pCB200, which was derived from the vector pBI121 (Clonetech) by replacing the kanamycin resistance (kan-r) gene with the E. coli BAR gene for resistance to the herbicide Liberty (Becker et al., Plant Mol. Biol. 20: 1195-1197 (1992)). The full-length ACS cDNA was cloned into pCB200 downstream of the CaMV 35S promoter in normal and opposite orientation for sense and antisense expression, respectively, 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, N.Y.) 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 selected by sowing seeds in sterile soil. Fourteen days later, the seedlings were sprayed with a solution of 0.5% glufosinate (w/v) (active ingredient of the herbicide Liberty). Non-transgenic plants died within about 7 days, whereas transgenic seedlings survived, were grown to maturity, and seeds were collected.
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 tested for resistance to the herbicide Liberty. 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 resistant to Liberty.
Two independent transgenic lines of ACS antisense plants were generated. The first line was characterized through the T3 generation and ACS enzyme activity levels as low as 20% of wild-type activity were observed. The second line was characterized through the T2 generation and ACS enzyme activity levels as low as 11% of wild-type activity were observed.
Twenty independent transgenic lines of ACS sense plants were generated. One line was characterized through the T3 generation, whereas all other lines were characterized through the T2 generation. ACS enzyme activity levels as high as 166% to 219% of wild-type activity were observed.
Thus, these data show that the expression of ACS, which generates acetyl CoA, can be increased and decreased.
Example 22
This example describes the alteration of pPDH levels in a plant using antisense nucleic acids.
Using the methods of Example 21, full-length pPDH E1α or pPDHE1β cDNA was cloned into the plant expression vector designated pCB200 downstream of the CaMV 35S promoter in opposite orientation from normal for antisense RNA expression. Two independent transgenic lines of PPDHE1α antisense plants and three independent transgenic lines of pPDHE1β antisense plants were generated and have been grown through the T1 generation. These plants will be analyzed in accordance with the methods of Example 21.
Example 23
This example describes the alteration of ALDH levels in a plant using sense and antisense nucleic acids.
Using the methods of Example 21, full-length ALDH cDNA was cloned into plant expression vectors in normal and opposite orientation for sense and antisense expression as follows:

    pCGN8641, napin promoter, kan-r gene (2 sense; 1 antisense)
    pCGN8643, napin promoter, kan-r gene (2 antisense; 1 sense)
    pCGN8640, CaMV 35S promoter, plant resistance gene bar (Liberty herbicide) (2 antisense; 1 sense)
    pCGN8644, CaMV 35S promoter, bar gene (2 sense; 1 antisense) pKMB (Mylne and Botella, Plant Molec. Biol. Reporter 16: 257-262 (1998)), CaMV 35S promoter, bar gene (3 sense)
    pSMB (Mylne and Botella (1998), supra), CaMV 35S promoter, bar gene (3 antisense).


A binary vector for plant transformation, pCGN5139, was constructed from pCGN1558 (McBride and Summerfelt, Plant Molecular Biology 14: 269-276 (1990)). The polylinker of pCGN1558 was replaced as a Hin dIII/Asp718 fragment with a polylinker containing unique restriction endonuclease sites, Asc I, Pac I, Xba I, Swa I, Bam HI and Not I. The Asp718 and Hin dIII restriction endonuclease sites are retained in pCGN5139.
A series of turbo binary vectors are constructed to allow for the rapid cloning of DNA sequences into binary vectors containing transcriptional initiation regions (promoters) and transcriptional termination regions.
The plasmid pCGN8618 was constructed by ligating oligonucleotides 5′-TCGAGGATCCGCGGCCGCAAGCTTCCTGCAGG-3′[SEQ ID NO: 31] and 5′-TCGACCTGCAGGAAGCTTGCGGCCGCGGATCC-3′[SEQ ID NO: 32] into Sal I/Xho I-digested pCGN7770. A fragment containing the napin promoter, polylinker and napin 3′ region was excised from pCGN8618 by digestion with Asp718I; the fragment was blunt-ended by filling in the 5′ overhangs with Klenow fragment then ligated into pCGN5139 that had been digested with Asp718I and Hin dIII and blunt-ended by filling in the 5′ overhangs with Klenow fragment. A plasmid containing the insert oriented so that the napin promoter was closest to the blunted Asp718I site of pCGN5139 and the napin 3′ was closest to the blunted Hin dIII site was subjected to sequence analysis to confirm both the insert orientation and the integrity of cloning junctions. The resulting plasmid was designated pCGN8622.
The plasmid pCGN8619 was constructed by ligating oligonucleotides 5′-TCGACCTGCAGGAAGCTTGCGGCCGCGGATCC-3′[SEQ ID NO: 33] and 5′-TCGAGGATCCGCGGCCGCAAGCTTCCTGCAGG-3′[SEQ ID NO: 34] into Sal I/Xho I-digested pCGN7770. A fragment containing the napin promoter, polylinker and napin 3′ region was removed from pCGN8619 by digestion with Asp718I; the fragment was blunt-ended by filling in the 5′ overhangs with Klenow fragment and then ligated into pCGN5139 that had been digested with Asp718I and Hin dIII and blunt-ended by filling in the 5′ overhangs with Klenow fragment. A plasmid containing the insert oriented so that the napin promoter was closest to the blunted Asp718I site of pCGN5139 and the napin 3′ was closest to the blunted Hin dIII site was subjected to sequence analysis to confirm both the insert orientation and the integrity of cloning junctions. The resulting plasmid was designated pCGN8623.
The plasmid pCGN8620 was constructed by ligating oligonucleotides 5′-TCGAGGATCCGCGGCCGCAAGCTTCCTGCAGGAGCT-3′[SEQ ID NO: 35] and 5′-CCTGCAGGAAGCTTGCGGCCGCGGATCC-3′[SEQ ID NO: 36] into Sal I/Sac I-digested pCGN7787. A fragment containing the d35S promoter, polylinker and tml 3′ region was removed from pCGN8620 by complete digestion with Asp718I and partial digestion with Not I. The fragment was blunt-ended by filling in the 5′ overhangs with Klenow fragment then ligated into pCGN5 139 that had been digested with Asp718I and Hin dIII and blunt-ended by filling in the 5′ overhangs with Klenow fragment. A plasmid containing the insert oriented so that the d35S promoter was closest to the blunted Asp718I site of pCGN5139 and the tml 3′ was closest to the blunted Hin dIII site was subjected to sequence analysis to confirm both the insert orientation and the integrity of cloning junctions. The resulting plasmid was designated pCGN8624.
pCGN8640 is a modification of pCGN8624. A 938 bp Pst I fragment isolated from transposon Tn7 which encodes bacterial spectinomycin and streptomycin resistance (Fling et al., Nucleic Acids Research 13(19):7095-7106 (1985)), a determinant for E. coli and Agrobacterium selection, was blunt ended with Pfu polymerase. The blunt-ended fragment was ligated into pCGN8624 that had been digested with Spe I and blunt-ended with Pfu polymerase. The region containing the Pst I fragment was sequenced to confirm both the insert orientation and the integrity of cloning junctions.
The spectinomycin resistance marker was introduced into pCGN8622 and pCGN8623 as follows. A 7.7 Kbp AvrII-SnaBI fragment from pCGN8640 was ligated to a 10.9 Kbp Avr II-Sna BI fragment from pCGN8623 or pCGN8622. The resulting plasmids were pCGN8641 and pCGN8643, respectively.
The plasmid pCGN8644 was constructed by ligating oligonucleotides 5′-GATCACCTGCAGGAAGCTTGCGGCCGCGGATCCAATGCA-3′[SEQ ID NO: 37] and 5′-TTGGATCCGCGGCCGCAAGCTTCCTGCAGGT-3′[SEQ ID NO: 38] into Bam HI-Pst I digested pCGN8640.
Transgenic plants are generated and analyzed according to the methods of Example 21, with the exception that transgenic plants generated with the vectors pCGN8641 and pCGN8643 were 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×Murashige and Skoog's salts (Sigma Chemical Co., St. Louis, Mo.), 1% sucrose, 1×Gamborg's vitamin (Sigma), 0.5 g/l 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, Wash.). 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, Ill.).
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 on 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.
Example 24
This example describes the alteration of PDC levels in a plant using sense and antisense nucleic acids.
Using the methods of Example 21, full-length PDC cDNA was cloned into plant expression vectors in normal and opposite orientation for sense and antisense expression as follows:

    pCGN8641, napin promoter, kan-r gene (1 sense; 1 antisense)
    pCGN8643, napin promoter, kan-r gene (1 sense)
    pCGN8640, CaMV 35S promoter, bar gene (1 sense)
    pCGN8644, CaMV 35S promoter, bar gene (1 sense)
    pKMB, CaMV 35S promoter, bar gene (1 antisense)
    pSMB, CaMV 35S promoter, bar gene (1 antisense).


Transgenic plants are generated and analyzed according to the methods of Examples 21 and 23.
Example 25
This example describes the alteration of ACL levels in a plant using sense and antisense nucleic acids.
Using the methods of Example 21, full-length ACL-A1 or ACL-B2 cDNA was cloned into plant expression vectors in normal and opposite orientation for sense and antisense expression as follows:

    pBI121 derivative plasmid, CaMV 35S promoter, kan-r gene (1 ACL-A1 sense; 1 ACL-B2 sense; 1 ACL-A1 antisense; 1 ACL-B2 antisense)
    pBI121 derivative plasmid, deleted CAC1 promoter (nucleotides −529 to +32 of the CAC1 promoter, wherein the nucleotides are numbered relative to the adenosine nucleotide of the ATG translation start codon, which is +1), kan-r gene (1 ACL-A1 sense; 1 ACL-B2 sense)
    pBI121 derivative plasmid, CaMV 35S promoter, kan-r gene (1 ACL-A1 (fused to plastid target sequence) sense).


All transgenic plants were selected upon the basis of kanamycin resistance as described in Example 23.
Transgenic plants comprising sense ACL-A1 under the control of the deleted CAC1 promoter were generated and have been grown through the T2 generation. Such plants exhibit an altered phenotype of very large leaves.
Transgenic plants comprising antisense ACL-A1 under the control of the CaMV 35S promoter were generated and have been grown through the T2 generation. Such plants exhibit two types of phenotypes. One type of phenotype comprises a much reduced plant body, shorter and thinner inflorescence stalks, smaller or non-opened flowers, reduced and occasionally early senescing petals, anthers with apparent problems of timing or dehiscence mechanisms, siliques, if present, are reduced, partially filled with seed or empty, smaller dessicated seeds within siliques, premature dehiscence within filled siliques, and very reduced leaves exhibiting anthocyanin production.
The other type of phenotype comprises smaller plants with milder versions of the characteristics described for the preceding phenotype, thinner, curled siliques with more easily differentiated external seed outlines, which often contain shriveled, dry seeds, and delayed dehiscence.
Other transgenic plants are generated and analyzed according to the methods of Examples 21 and 23.
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

1. A method of altering the level of an enzyme 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 nucleic acid molecule selected from the group consisting of (i) a coding sequence for a plant plastidic ACS or (ii) a nucleic acid molecule comprising or encoding a ribozyme to an RNA molecule transcribed from a coding sequence of (i), wherein said vector comprising a nucleic acid molecule of (i) increases the level of said enzyme in said plant cell, plant tissue, plant organ or plant, and wherein said vector comprising a nucleic acid molecule of (ii) decreases the level of said enzyme in said plant cell, plant tissue, plant organ or plant.

2. The method of claim 1, wherein said enzyme is plastidic ACS.

3. The method of claim 1, wherein the alteration of said enzyme results in an alteration of the level of acetyl CoA in said plant cell, plant tissue, plant organ or plant.

4. The method of claim 2, wherein the alteration of said enzyme results in an alteration of the level of acetyl CoA in said plant cell, plant tissue, plant organ or plant.

5. A plant cell, a plant tissue, a plant organ or a plant in which the level of plastidic ACS has been altered in accordance with the method of claim 1.

6. A plant cell, a plant tissue, a plant organ or a plant in which the level of plastidic ACS has been altered in accordance with the method of claim 2.

7. A plant cell, a plant tissue, a plant organ or a plant in which the level of acetyl CoA has been altered in accordance with the method of claim 3.

8. A plant cell, a plant tissue, a plant organ or a plant in which the level of acetyl CoA has been altered in accordance with the method of claim 4.