CA2263186A1 - Transgenic plant cells and plants with modified acetyl-coa formation - Google Patents

Abstract

The invention concerns transgenic plant cells and plants having increased acetyl-coA hydrolase activity. The increased coA hydrolase activity is achieved by introducing and expressing a DNA sequence which codes for an acetyl-coA hydrolase, preferably a deregulated or unregulated acetyl-coA hydrolase, in plant cells. The invention further concerns processes and recombinant DNA molecules for producing plant cells and plants having increased acetyl-coA hydrolase activity.

Description

MAX-PLANCK-GESELLSCHAFT. . .
Our ref.: B 2314 PCT

Transgenic plant cells and plants with modified acetyl CoA production The present invention relates to transgenic plant cells and plants with a modified acetyl CoA metabolism and with a modified ability to produce and utilize acetyl CoA (acetyl coenzyme A) as compared with non-transformed plants. The modification in the ability to produce and utilize acetyl CoA is brought about by introducing and expressing in plant cells a DNA sequence which encodes preferably a deregulated or unregulated acetyl CoA hydrolase.
The invention furthermore relates to the use of DNA sequences encoding an acetyl CoA hydrolase for increasing the acetyl CoA hydrolase activity in plant cells, particularly for producing transgenic plant cells and plants having a modified ability to produce and utilize acetyl CoA.

Due to the constantly increasing demand in food, which is a result of the incessantly growing world population, it is one of the objectives of research inthe field of biotechnology to try to increase the yield of useful plants. One possibility of achieving this object is to specifically modify the metabolism ofplants by way of genetic engineering. The subjects of these modifications are, for example, the primary processes of photosynthesis that lead to CO2 fixation, the transport processes that are involved in the distribution of the photoassimilates within the plant, as well as metabolic pathways leading to the synthesis of storage substances such as starch, proteins, fats, oils, rubbersubstances or of secondary metabolites such as flavonoids, steroids, isoprenoids (e.g., flavors), pigments, or polyketides (antibiotics), or of plantpathogen defense substances. While many uses deal with the steps which either lead to the production of photoassimilates in leaves (cf. also EP-A 0 466 995) or with the production of polymers such as starch or fructans in the storage organs of transgenic plants (e.g., WO 94/04692), there are no promising approaches yet which describe which modifications have to be introduced into the primary metabolic pathways in order to achieve a modification in the ability to produce and utilize acetyl CoA. A modification inthe ability to produce and utilize acetyl CoA is important, e.g., for all those processes in the plant for which major amounts of acetyl CoA are required.
This is, for example, true for many biochemical processes in plant cells in which acetyl CoA is involved as substrate or degradation product. A
modification of the acetyl CoA production rate is of particular importance for the production of starch, proteins, fats, oils, rubber substances, or of secondary metabolites such as flavonoids, steroids, isoprenoids (flavors, pigments) or polyketides (antibiotics). Since plants would be suitable for producing several of the above-mentioned substances on a large scale due to various properties they possess, there is a demand in plants in which the production and distribution of acetyl CoA in the cells is modified in such a waythat the production of the substances described above is influenced.

It is thus the problem underlying the present invention to provide plant cells and plants with a modified ability to produce and utilize acetyl CoA as well as processes for producing said plant cells and plants.

This problem is solved by providing the embodiments characterized in the patent claims.

Thus, the present invention relates to transgenic plant cells with a modified acetyl CoA metabolism which exhibit an elevated acetyl CoA hydrolase activity as compared to wild-type cells, i.e., non-transformed plants, due to the expression of an exogenous DNA sequence encoding a protein with acetyl CoA hydrolase activity. The expression of such a DNA sequence leads to an increase in intracellular acetyl CoA hydrolase activity in the transgenic plant cells. Acteyl CoA hydrolases are enzymes catalyzing the following reaction:

Acetyl CoA ~ acetate + HSCoA.

It was surprisingly found that it is possible to increase the acetyl CoA
hydrolase activity in various compartments of plant cells, thereby allowing to influence the intracellular distribution of metabolites. This finding is all themore so surprising since acetyl CoA is one of the primary metabolites in plant and animal cells whose concentration is strictly regulated (Randall and Miernyk, Methods in Plant Biochemistry Vol. 3 [ISBN 0-12~61013-7]).
Influencing the intracellular acetyl CoA distribution should therefore dramatically affect the viability of the cells. For example, there are hints that the expression of the acetyl CoA hydrolase from yeast has a lethal effect in E. coli. The present invention is based on the finding that it is factually possible to inrease the acetyl CoA hydrolase activity in plant cells and that such an increase results in advantageous properties of the plant cells. It was found, e.g., that the inuease in the acetyl CoA hydrolase activity in the mitochondria in the leaves of transgenic plants results in an increase in the concentration of soluble sugars such as glucose, fructose and sucrose, as well as starch while the concentration of fatty acids is simultaneously reduced.That means that an increase in the mitochondrial acetyl CoA hydrolase activity allows a modification of the partitioning of photoassimilates in the cells. The acetyl CoA hydrolase activity in the cells according to the inventionis preferably elevated by at least 50% and particularly preferred by 100% as compared to non-transformed cells. Particularly advantageous is an increase in enzyme activity by more than 150% as compared to non-transformed cells.
The increase in acetyl CoA hydrolase activity results in an elevated concentration of acetate. Unlike acetyl CoA, acetate may penetrate cellular membranes without regulation. Thus, it is available in other cellular compartments in higher concentration as substrate for the acetyl CoA
synthesis catalyzed by the acetyl CoA synthetase. This means that it is possible to modify the intracellular distribution of acetyl CoA by increasing the acetyl CoA hydrolase activity in one compartment, thereby influencing the metabolic flow in various pathways of biosynthesis.

In a preferred embodiment, the present invention relates to transgenic plant cells in which the acetyl CoA hydrolase activity in the mitochondria is elevated. In plant cells, biosynthesis of acetyl CoA in the mitochondria takes place by the reaction of pyruvate catalyzed by the pyruvate dehydrogenase multienzyme complex. An increase in acetyl CoA hydrolase activity in the mitochondria results in an elevated concentration of acetate which may diffuse across cellular membranes to other compartments, e.g., to the cytosol.
There, it may be utilized for the synthesis of acetyl CoA, e.g., by the acetyl CoA synthetase.

In another preferred embodiment, the transgenic plant cells according to the invention therefore display an elevated activity of acetyl CoA synthetase in thecytosol. Said enzyme catalyzes the following reaction:

Acetate + HSCoA ~ acetyl CoA + AMP +PPj The acetyl CoA increasingly produced in the cytosol may, for example, be utilized for a greater synthesis of isoprenoids via mevalonic acid and isopentenyl pyrophosphate (Bach, Lipids 30 (1995), 191-202).

In another preferred embodiment, the acetyl CoA hydrolase activity is elevated in the cytosol of the transgenic plant cells, thereby again achieving an elevated acetate concenlralion. The latter may have the result that there is more acetate available in the plastids and is converted there to acetyl CoA, thereby providing more acetyl CoA as substrate, e.g., for fatty acid biosynthesis or isoprenoid synthesis.

In a particularly preferred embodiment, the transgenic plant cells according to the invention which display an elevated acetyl CoA hydrolase activity in the mitochondria or the cytosol also display an elevated activity of the acetyl CoA
synthetase in the plastids, thereby promoting the shift of acetate to the plastids and its conversion to acetyl CoA. The latter is then available to a higher extent for, e.g., fatty acid biosynthesis.
The reduction of the acetyl CoA synthetase activity in the cytosol is in principle also possible.

In another preferred embodiment, the above-described plant cells display a reduced citrate synthase activity in the mitochondria. Said enzyme catalyzes the following reaction:

Acetyl CoA + oxaloacetate ~ citrate + HSCoA

The reduction of the activity of said enzyme utilizing acetyl CoA as substrate for the citrate synthesis makes more acetyl CoA available for the reaction catalyzed by acetyl CoA hydrolase, leading to an increased production of acetate.

Another preferred embodiment of the present invention provides that in the above-described plant cells according to the invention the activity of the citrate synthase is elevated in the mitochondria or the cytosol.
Such an increase in the activity of the citrate synthase may lead to a modification of the flow of metabolites in direction of the acetyl CoA to a certain subcellular compartment and may specifically cause an increase in biosynthesis of fatty acids or lipids.

In another preferred embodiment of the present invention, the above-described plant cells of the invention also display a reduced activity of the ATP citrate Iyase in the cytosol. Said enzyme catalyzes the following reaction:

Citrate+ HSCoA + ATP ~ acetyl CoA + AMP + PPi + oxaloacetate.

Such a reduction may lead to an increase in the metabolite flow of citrate in direction of acetyl CoA which may cause a backpressure of metabolites in the citric acid cycle. Therefore, it would be possible that acetate is increasingly produced via the endogenous acetyl CoA hydrolase, which may increase the production of lipids and/or terpenoids.

Another embodiment of the present invention provides that the plant cells display an elevated ATP citrate Iyase activity in the cytosol.
Such an increase may result in an elevated production of cytosolic acetyl CoA, which may lead to an increased synthesis of isopentenyl pyrophosphate (IPP) and thus to an elevated production of terpenoids.

The above-described transgenic plant cells display a modified ability to produce and utilize acetyl CoA as compared with non-transformed plants due to the modified enzyme activities described above. This ability may, e.g., be detected by determining the modified amounts or ratios of metabolic end products and metabolic intermediates as described in the examples. In particular, plant cells according to the invention can be produced which display modified amounts of isoprenoids, steroids, pigments, isoprenoids, flavonoids, hormones, fats, oils, proteins, rubber substances, polyketides or substances that are involved in the plant pathogen defense, or their content of soluble sugars such as glucose, fructose and sucrose, as well as their starch content.
Such cells may in turn be advantageous as starting material for other uses.
For example, these cells may serve for the heterologous expression of other genes with the purpose of an increased synthesis of economically relevant substances. DNA sequences may be introduced which encode the enzymes required for the synthesis of polyhydroxy alkanoic acids (e.g., PHB and PHA).
In this manner, the increasingly produced acetyl CoA may be utilized in plants to synthesize such acids which are of great economic importance. Other examples of economically relevant substances are polyketides, flavors, caoutchouc, alkaloids, isoprenoids, etc.
Of specific importance is the possibility to direct the flow of the photoassimilates deposited in the seeds or organs in direction of the production of sugars, starch, fats, pigments, isoprenoids, polyketides, steroids, flavonoids, rubber substances, substances that are involved in the plant pathogen defense, proteins, and polymers such as polyhydroxy alkanoic acids (cf., e.g., Poirier et al., Bio/Technology 13 (1995), 142-150) by expressing an acetyl CoA hydrolase in oil-storing tissues of a plant such as the endosperm or the cotyledons of the seeds or other oil-storing tissues. A
general advantage of the cells according to the invention is the possibility to influence the partitioning of metabolites, particularly of acetyl CoA, the content of metabolic end products such as starch and fats, the content and composition of secondary metabolites, the energy balance and the content and composition of amino acids in the cells.

In a preferred embodiment of the present invention, the transgenic plant cells are cells of oil-storing tissues, e.g., of the endosperm or the cotyledons of the seeds or other oil-storing organs. Preferably, these cells, when compared to the respective cells of non-transformed plants, display a content of fats that is elevated by at least 3%, preferably by at least 5% and particularly preferred by at least 7%.

The increase in acetyl CoA hydrolase activity in the cells according to the invention is preferably brought about by introducing and expressing DNA
sequences encoding an acetyl CoA hydrolase. These DNA sequences which encode a protein having the enzymatic activity of an acetyl CoA hydrolase may be both prokaryotic, particularly bacterial, and eukaryotic DNA
sequences, i.e., DNA sequences from plants, algae, fungi or animal organisms, or sequences which encode acetyl CoA hydrolases from those organisms.

In a preferred embodiment of the invention, the DNA sequences encoding an acetyl CoA hydrolase are sequences encoding enzymes which compared to acetyl CoA hydrolases normally occurring in plant cells are deregulated or unregulated. Deregulated means that these enzymes are not regulated in the same manner as the acetyl CoA hydrolase enzymes normally produced in non-modified plant cells. These enzymes specifically are subject to other regulatory mechanisms, i.e., they are not inhibited to the same extent by the inhibitors present in the plant cells or allosterically regulated by metabolites.
Deregulated preferably means that the enzymes exhibit a higher activity than acetyl CoA hydrolases endogenously expressed in plant cells. Wlthin the present invention, unregulated means that the enzymes are not subject to any regulation in plant cells.
The enzymes encoded by these sequences may be both known naturally occurring enzymes exhibiting a regulation modified by various substances, and enzymes produced by mutagenesis of DNA sequences which encode known enzymes from bacteria, algae, fungi, animals or plants.

In a specifically preferled embodiment of the present invention, the DNA
sequences used encode proteins with the enzymatic activity of an acetyl CoA
hydrolase from fungi, particularly from fungi of the genus Saccharomyces.
Preferably, DNA sequences are used encoding an acetyl CoA hydrolase from Saccharomyces cerevisiae. Such sequences are known and have been described earlier (cf. Lee et al., Journal of Biological Chemistry 265 (1990), 7413-7418 (accession no. M31036)). In order to ensure the localization of the acetyl CoA hydrolase in mitochondria of the plant cells, the DNA sequences encoding mitochondria targeting sequences have to be fused to the coding region of the acetyl CoA hydrolase. Such sequences are known, for example, from Braun et al. (EMBO J. 11 (1992), 3219-3227).
In addition to the above-mentioned DNA sequence from Saccharomyces cerevisiae further DNA sequences are known which encode proteins having the enzymatic activity of an acetyl CoA hydrolase, for example from Neurospora crassa (cf. EMBL accession no. M31521; Marathe et al., Molecular and Cellular Biology 10 (1990), 2638-2644) and which due to their properties may also be used for producing the plant cells according to the invention. For this purpose, it must be taken care that the protein is produced in the mitochondria or the cytosol of the plant cell. Techniques for modifying such DNA sequences in order to ensure the localization of the synthetized enzymes in the mitochondria and in the cytosol of the plant cells are known to the person skilled in the art. Should the acetyl CoA hydrolases contain sequences that are necessary for the secretion or for a specific subcellular localization, e.g., for the localization in the extracellular space or the vacuole, the respective DNA sequences have to be deleted. Also,DNA sequences encoding an acetyl CoA hydrolase may be isolated from any organism by using the above-mentioned DNA sequences that are already known. The person skilled in the art is familiar with methods for isolating and identifyingsuch DNA sequences, for example the hybridization with known sequences or by polymerase chain reaction using primers that are derived from known sequences.

The enzymes encoded by the identified DNA sequences are then examined for their enzymatic activity and regulation.
The proteins encoded by the DNA sequences may then be further modified with respect to their regulatory properties by introducing mutations and modifications according to techniques well-known to the person skilled in the art in order to obtain enzymes that are deregulated or unregulated as compared to acetyl CoA hydrolases naturally occurring in plants.

The increase in acetyl CoA synthase, citrate synthase or ATP-citrate Iyase activity is preferably brought about in the plant cells according to the invention by introducing and expressing DNA sequences encoding such enzymes.
These sequences may be sequences encoding such enzymes from prokaryotic, particularly bacterial, or from eukaryotic organisms such as plants, algae, fungi or animals.

In a preferred embodiment, such enzymes are deregulated or unregulated enzymes, as has been explained earlier in context with the acetyl CoA
hydrolase.
The enzymes encoded by these sequences may be both known naturally occurring enzymes exhibiting a regulation modified by various substances, and enzymes produced by mutagenesis of DNA sequences which encode known enzymes from bacteria, algae, fungi, animals or plants.

DNA sequences encoding acetyl CoA synthases from various organisms havebeen described. For animal organisms, examples are known, e.g., from Macropus engenii, human, Caenorhabditis elegans and Drosophila melanogaster (see, e.g., GenEMBL data bank accession nos. L15560, D16350, Z66495 and Z46786).

In a particularly preferred embodiment of the present invention, the DNA
sequences encode an acetyl CoA synthetase with the biological properties of an acetyl CoA synthase from fungi, particularly from those of the genus Saccharomyces, and particularly preferred from Saccharomyces cerevisiae.
Such sequences are, e.g., available under the GenEMBL data bank accession nos. Z47725, M94729, L09598, X56211 for Saccharomyces cerivisiae particularly under X76891. It is also possible to use DNA sequences encoding bacterial acetyl CoA synthetases. Such DNA sequences are available, e.g., under accession nos. M97217, M87509 or M63968.

DNA sequences encoding a citrate synthase are known from various organisms. Sequences encoding plant citrate synthases are known, e.g., for Arabidopsis thaliana (GenEMBL data bank accession no. X17528; Unger et al., Plant Mol. Biol. 13 (1989), 411418), as well as for tobacco, potato and sugar beet (see WO 95/24487). Furthermore, sequences are known that encode animal citrate synthases, e.g., from pig (accession no. M21197, Evans et al., Biochemistry 27 (1988), 46804686). Preferably, sequences are used which encode a citrate synthase with the biological properties of a citratesynthase from bacteria, particularly from E. coli, or from fungi, particularly from Saccharomyces cerevisiae. Various sequences encoding the citrate synthases from bacteria are available, e.g. under GenEMBL data bank ~ccession nos.: M33037, Z70021, M74818, Z70017, Z70009, Z70016, L38987, Z70014, Z70022, Z70019, Z70018, Z70020, Z70012, Z70010, Z70011, Z70013, Z70015, M36338, L33409, X66112, X60513, Z73101, M29728, M17149, L41815, Z34516, M73535, L14780, X55282, L75931 and D90117. A sequence that is preferably used is that published in Ner et al.
(Biochemistry 22 (1983), 5243-5249) which encodes a citrate synthase from E. co/i. Sequences encoding citrate synthases from E. co/i are available, e.g., under accession nos. M28987 and M28988 (see also Wilde et al., J. Gen Microbiol. 132 (1986), 3239-3251). Sequences encoding the citrate synthases from fungi are available under accession nos. D63376 and D69731, those from S. cerevisiae particularly under accession nos. Z11113, Z48951, Z71255, M54982, X88846 and X00782. The latter is preferably used.

DNA sequences encoding an ATP citrate Iyase are known, e.g., from rats (Elshourbagy et al., J. Biol. Chem. 265 (1990), 1430-1435), human (Elshourbagy et al., Eur. J. Biochem. 204 (1992), 491499), C. elegans (Wilson et al., Nature 368 (1994), 32-38) and Arabidopsis thaliana (EMBL
accession nos. T13771, Z18045, Z25661 and Z26232).

With respect to the localization of the respective enzyme in the desired compartment of the plant cell the same applies as has already been said in context with the acetyl CoA hydrolase.

The reduction of the activity of the citrate synthase or the ATP citrate Iyase in the plants according to the invention may be brought about by methods .. . ..

known to the person skilled in the art, e.g., by expression of an antisense RNA, a specific ribozyme or by way of a cosuppression effect.

In order to ensure the expression of the DNA sequences encoding the above-described enzymes in plant cells, they can be basically put under the control of any promoter functional in plant cells. The expression of said DNA
sequences can generally take place in any tissue of a plant regenerated from a transformed plant cell according to the invention and at any time. However, it preferably takes place in those tissues in which a modified ability to produce and utilize acetyl CoA is advantageous either for the growth of the plant or forthe production of the ingredients within the plant. Therefore, promoters ensuring a specific expression in a certain tissue, at a certain stage of development of the plant or in a certain organ of the plant seem to be especially suitable. Preferably, the DNA sequences are under the control of promoters that ensure a seed-specific expression. Thus, in the case of starch-storing plants such as maize, wheat, barley or other cereals, the ability to produce and utilize acetyl CoA in the seeds and the synthesis of seed ingredients is modified.

In a preferred embodiment, promoters are used for increasing the fatty acid biosynthesis on the basis of an elevated acetyl CoA content in the seeds of oil-producing plants such as rape, soy bean, sun flower and oil palm which are specifically active in the endosperm or in the cotyledons of nascent seed, such as the phaseolin promoter from Phaseolus vulgaris, the USP promoter from Vicia faba or the HMG promoter from wheat.
According to the invention it is also advantageous to use promoters for expression of the DNA sequences that are active in storage organs such as tubers or roots, e.g., in the storage root of the sugar beet or in the tuber of the potato. In this case, for example, upon expression of the DNA sequences encoding an acetyl CoA hydrolase, the biosynthesis pathways are redirected in the sense that more sugar or starch is produced, and the production and utilization of acetyl CoA is modihed in direction of a fatty acid biosynthesis.
Also expression of the DNA sequences may take place under the control of promoters that are activated specifically at the time of flower induction, or that are activated upon flower formation, or that are active in tissues that are necessary for the flower induction. Likewise, promoters may be used that are activated only at a point of time controlled by external influences such as light, temperature, chemical substances (see, e.g., WO 93/07279). For increasing the export rate of photoassimilates from the leaf, e.g., promoters are of interest that exhibit a companion cell specific expression. Such promoters are known (e.g., the promoter of the rolC gene from Agrobacterium rhizogenes).
The DNA sequences encoding the above-described enzymes, in addition to a promoter, are preferably also linked with DNA sequences ensuring a further increase of transcription, for example, so-called enhancer elements, or with DNA sequences that are located in the transcribed region and ensure a more efficient translation of the synthetized RNA into the corresponding protein.
Such regions may be derived from viral genes or appropriate plant genes or may be synthetically produced. The may be homologous or heterologous with respect to the promoter used. Advantageously, the coding DNA sequences are also linked with 3'-nontranslated DNA sequences that ensure transcription termination and polyadenylation of the transcript. Such sequences are known and have been described in the art, for example those of the octopin synthase gene from Agrobacterium tumefaciens. These sequences are freely interchangeable.

The DNA sequences which are introduced and expressed according to the invention in plant cells are present in the plant cells according to the invention preferably stably integrated into the genome. In addition to the above-described modified enzymatic activities the transgenic plant cells according to the invention can be also distinguished from non-transformed plant cells by the fact that they contain a foreign DNA stably integrated into the genome the expression of which effects modification of the acetyl CoA hydrolase activity and optionally a further of the above-described enzymatic activities. Foreign DNA means in this context that the DNA is either heterologous with respect to the transformed plant species, or the DNA, if it is homologous to the plant species, is localized at a locus in the genome where it does not occur in non-transformed cells. This means that the DNA is localized in a genomic surround in which it does not naturally occur. Furthermore, the foreign DNA
usually is characterized in that it is recombinant, i.e., consists of several components which do not naturally occur in this combination.

The transgenic plant cells according to the invention may basically be cells of any plant species. Of interest are both cells of monocotyledonous and dicotyledonous plant species, particularly cells of starch-storing or agricultural useful plants such as rye, oats, barley, wheat, potatoes, maize, rice, peas, sugar beet, tobacco, cotton, wine, tomatoes, etc., or cells from ornamental plants.

In a preferred embodiment, the plant cells are cells of oil-storing useful plants such as rape, sun flower, oil palm or soy bean. Rape is particularly preferred.
Another subject matter of the present invention are transgenic plants containing transgenic plant cells according to the invention. Such plants may, for example, be produced by regeneration from plant cells according to the invention according to methods known to the person skilled in the art.

Plants containing cells according to the invention preferably exhibit at least one of the following features:
(a) a reduced or elevated content of fatty acids in the leaf tissue or in the seed tissue as compared to wild-type plants;
(b) an elevated content of soluble sugars in the leaf tissue as compared to wild-type plants;
(c) an elevated content of starch in the leaf tissue as compared to wild-type plants;
(d) reduced growth;
(e) formation of two or more shoots;
(f) modified leaf coloration.

Another subject matter of the invention is the propagation material of plants according to the invention which contains cells according to the invention.
Examples thereof are cuttings, seeds, fruits, root stocks, tubers, seedlings, etc.

Another subject matter of the invention are recombinant DNA molecules containing the following elements:
(a) a promoter functional in plant cells, and (b) a DNA sequence encoding a protein having the enzymatic activity of an acetyl CoA hydrolase and being linked to the promoter such that it may be transcribed in plant cells into a translatable RNA.

The transfer of the DNA molecules containing the DNA sequences encoding one of the above-described enzymes is carried out according to methods known to the person skilled in the art, preferably using plasmids, particularly those plasmids ensuring a stable integration of the DNA molecule into the , . . ....... . .. . . . . . .. . ... . .

genome of transformed plant cells, for example binary plasmids or Ti plasmids of the Agrobacterium tumefaciens system. Besides the Agrobacterium system there are other systems available for introducing DNA molecules into plant cells, for example the so-called biolistic method or the transformation of protoplasts (cf. Willmitzer L. (1993), Transgenic Plants, Biotechnology 2; 627-659 for an overview). Methods for transforming monocotyledonous and dicotyledonous plants have been described in the literature and are known to the person skilled in the art.

Finally7 the present invention relates to the use of DNA sequences encoding a protein with the enzymatic activity of an acetyl CoA hydrolase for expression in plant cells in order to increase the acetyl CoA hydrolase activity in plant cells. Specifically, the invention relates to the use of such DNA sequences for producing transgenic plant cells which compared to non-transformed plant cells have a modified ability to produce sugars, starch, fats, pigments, isoprenoids, polyketides, steroids, flavonoids, rubber substances, substances that are involved in the plant pathogen defense, proteins and/or polymers such as polyhydroxy alkanoic acids. The acetyl CoA hydrolase activity is preferably increased in the mitochondria or in the cytosol of the plant cells.

Fig. 1 shows a schematic diagram of the 14.39 kb plasmid Bin-mHy-lnt. The plasmid contains the following fragments:
A = Fragment A (528 bp) contains the EcoRI - Asp718 fragment of the promoter region of the 35S promoter of the Cauliflower Mosaic Virus (nucleotides 6909 to 7437) (Frank et al., Cell 21 (1980), 285-294).
B = Fragment B (109 bp) comprises a DNA fragment with the coding region of the mitochondrial target sequence of the protein of the matrix processing peptidase (MPP) from potato (Braun et al., EMBO J. 11 (1992), 3219-3227 (accession no. X66284)).
C = Fragment C (189 bp) comprises a DNA fragment of intron PIV2 from plasmid p35S GUS INT (Vancanneyt et al., Mol. Gen. Genet.
220 (1990), 245-250).
D' = Fragment D' (170 bp) comprises a DNA fragment with the 5' region of the coding region of the acetyl CoA hydrolase gene (Lee et al., Journal of Biological Chemistry 265 (1990), 7413-7418), nucleotides 614 to 784 (accession no. M31036).

D" = Fragment D" (1420 bp) comprises a DNA fragment with the coding region of the acetyl CoA hydrolase gene (Lee et al., Joumal of Biological Chemistry 265 (1990), 7413-7418), nucleotides 785 to 2194 (accession no. M31036).
E = Fragment E (192 bp) comprises the polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid pTi-ACH5, nucleotides 11749-11939 (Gielen et al., EMBO J. 11 (1984), 3219-3227).

Fig. 2 shows a schematic diagram of the 14.28 kb plasmid Bin-Hy-lnt. The plasmid contains following fragments:
A = Fragment A (528 bp) contains the EcoRI - Asp718 fragment of the promoter region of the 35S promoter of the cauliflower mosaic virus (Nucleotide 6909 to 7437) (Frank et al., Cell 21 (1980), 285-294).
C = Fragment C (189 bp) comprises a DNA fragment of intron PIV2 from plasmid p35S GUS INT (Vancanneyt et al., Mol. Gen. Genet.
220 (1990), 245-250).
D' = Fragment D' (170 bp) comprises a DNA fragment with the 5' region of the coding region of the acetyl CoA hydrolase gene (Lee et al., Journal of Biological Chemistry (1990) 265, 7413-7418), nucleotides 614 to 784 (accession no. M31036).
D" = Fragment D" (1420 bp) comprises a DNA fragment with the coding region of the acetyl CoA hydrolase gene (Lee et al., Journal of Biological Chemistry (1990) 265, 7413-7418), nucleotides 785 to 2194 (accession no. M31036).
E = Fragment E (192 bp) comprises the polyadenylation signal of gene 3 of the T-DNA of Ti plasmid pTi-ACH5, nucleotides 11749-11939 (Gielen et al., EMBO J. 11 (1984), 3219-3227).

Fig. 3 shows a Western-Blot for detection of the expression of the acetyl CoA
hydrolase from Saccharomyces cerevisiae in transgenic tobacco leaves.

Fig. 4 shows three transgenic MB-Hy1 lines as compared to a control plant (left).

Fig. 5 shows leaves of the transgenic MB-Hy1 line 39 Fig. 6 shows leaves of a control plant Fig. 7 shows a plant of a transgenic MB-Hy1 line with flowers Fig. 8 shows a control plant with flowers Fig. 9 shows a schematic diagram of the 14. 25 kb plasmid pTCSAS
A = Fragment A (528 bp) contains the EcoRI - Asp718 fragment of the promoter region of the 35S promoter of the Cauliflower Mosaic Virus (nucleotides 6909 to 7437) (Frank et al., Cell 21 (1980), 285-294).
. B = Fragment B (1747 bp) comprises a DNA fragment with the coding region of the citrate synthase gene from tobacco in reverse orientation (nucleotides 1 to 1747) (accession no. X84226).
C = Fragment C (192 bp) comprises the polyadenylation signal of gene 3 of the T-DNA of Ti plasmid pTi-ACH5, nucleotides 11749-11939 (Gielen et al., EMBO J. 11 (1984), 3219-3227).

Methods 1. Cloning methods For cloning in E coli, the vectors pUC9-2, pA7 (von Schaewen, A.
(1989) Dissertation, Freie Universitat Berlin) and pAM (see Example 1) were used. For plant transformation, the gene constructs were cloned into the binary vector pBinAR-Hyg (deposition no.: DSM 9505; date of deposition: October20, 1994).

2. Bacterial strains For the pUC vectors and for the pBinAR Hyg constructs, the E coli strain DH5a (Bethesda Research Laboratories, Gaithersburg, USA) was used.

The transformation of the plasmids in the potato plants was carried out using the Agrobacterium tumefaciens strain C58C1 pGV2260 (Deblaere et al., Nucl. Acids Res. 13 (1985), 4777-4788).

3. Transformation of Agrobacterium tumefaciens The transfer of the DNA was carried out by direct transformation according to the method by Hofgen and Willmitzer (Nucleic Acids Res.
16 (1988), 9877). The plasmid DNA of transformed Agrobacteria was isolated according to the method by Birnboim and Doly (Nucleic Acids Res. 7 (1979), 1513-1523) and was analyzed gel electrophoretically after suitable restriction digestion.

4. Transformation of tobacco or rape The transformation of tobacco was carried out according to the method described in Rosahl et al. (EMBO J. 6 (1987), 1155-1159).

The transformation of rape (Brassica napus) was carried out in analogy to the method described in Bade and Damm in "Gene Transfer to Plants" (SpringerVerlag Heidelberg (1995), pp. 30-38).

5. Transformation of potatoes Ten small leaves of a potato sterile culture (Solanum tuberosum L.cv.
Désirée) were wounded with a scalpel and placed in 10 ml MS medium (Murashige and Skoog, Physiol. Plant. 15 (1962), 473) containing 2%
sucrose which contained 50 ~l of a selectively grown overnight culture of Agrobacterium tumefaciens. After gently shaking the mixture for 3-5 minutes it was further incubated in the dark for 2 days. For callus induction, the leaves were placed on MS medium containing 1.6%
glucose, 5 mg/l naphthyl acetic acid, 0.2 mg/l benzyl aminopurine, 250 mg/l claforan, 3 mg/l hygromycin, and 0.80% Bacto Agar. After incubation at 25~C and 3,000 lux for one week the leaves were placed for shoot induction on MS medium containing 1.6% glucose, 1.4 mg/l zeatin ribose, 20 mg/l naphthyl acetic acid, 20 mg/l giberellic acid, 250 mg/l claforan, 3 mg/l hygromycin and 0.80% Bacto Agar.

6. Plant cultivation Potato plants were cultivated in a greenhouse under the following conditions:

Light period 16 hrs at 25,000 lux and 22~C
Dark period 8 hrs at 1 5~C
Humidity 60%.

Tobacco plants were cultivated in a greenhouse under the following conditions:
Light period 14 hrs at 10,000 lux and 25~C

Dark period 10 hrs at 20~C
Humidity 65%.

7. Detection of the acetyl CoA hydrolase activity in leaves of tobacco and potato plants For detecting the acetyl CoA hydrolase activity in tobacco and potato plants, leaf samples were homogenized in extraction buffer (50 mM
Hepes-KOH pH 7.5; 5 mM MgCI2; 1 mM EDTA; 1 mM EGTA; 10 mM
DTT; 10% (vol./vol.) glycerol; 0.1% (vol./vol.) Triton X-100). After centrifugation, the cell-free extracts were used for measuring the enzymatic activity.

Reaction buffer: 100 mM Na phosphate, pH 7.2 [1-14C]Acetyl-CoA 0.5 ~Ci /100 ~l Reaction volume: 105 ,ul Reaction temperature: 30~C
Time of measurement: 2, 4, 6, 10 min Principle:
DE81 filters (ion exchanger) were used; HSCoA and [1-14C]acetyl CoA
bind to each other, while [1-14C] is eluted when washing the filters (2 times 5 min) with 2% acetic acid. For determining the acetyl CoA

hydrolase activity, the radioactivity remaining on the filters was detected by scintillation measurement and comparison with calibration curves.
(cf. Roughan, P.G. et al., Analytical Biochemistry 216 (1994), 77-82) 8. Western blot analysis for detecting the expression of acetyl CoA
hydrolase in leaves For detecting the acetyl CoA hydrolase in tobacco and potato leaves, leaf samples were homogenized in extraction buffer (50 mM Hepes-KOH
pH 7.5; 5 mM MgCI2; 1 mM EDTA; 1 mM EGTA; 10 mM DTT; 10%
(vol./vol.) glycerol; 0.1% (vol./vol.) Triton X-100). After centrifugation aliquots (10 ,ug protein) of the cell-free extracts were used for a Westem blot. Western blot analyses were carried out using a polyclonal antibody directed to the acetyl CoA hydrolase from Saccharomyces cerevisiae as described by Landschutze et al. (EMBO J. 14 (1995), 660466).

9. Detection of metabolic intermediates in tobacco leaves Sucrose, glucose, fructose and starch were detected spectrophotometrically by coupled enzymatic reactions according to Stitt et al. (Methods in Enzymology, 174, 518-552).

a) Detection of sucrose. ~lucose and fructose Three leaf disks with a diameter 1.1 cm each were extracted for 90 min at 70~C with 500 1~1 80% ethanol each in water. After separating the solid from the liquid phase by centrifugation the liquid phase was removed and used to measure the soluble sugars.
The reaction buffer contained:
100 mM imidazol pH 6.9;
5 mM MgCI2;
2 mM NADP
1 mMATP
2 U/ml glucose4-phosphate dehydrogenase Detection of G6P: + 1.4 U/ml hexokinase Detection of F1 P: + 1.4 U/ml phosphoglucoisomerase Detection of G1 P: + 2.0 U/ml phosphoglucomutase Measurement at 30~C with 50 ,ul extract.

(G6P = glucose-6-phosphate; F1 P = fructose-1 -phosphate;
G1 P = glucose-1 -phosphate) b) Detection of starch Three leaf disks with a diameter 1.1 cm each were extracted for 90 min at 70~C with 500 ,ul 80% ethanol each in water. After separating the solid from the liquid phase by centrifugation the liquid phase was removed, the solid product was washed twice with 80% ethanol in water.
The pellet was extracted for one hour at 95~C with 400 ,ul 0.2 N
NaOH. It was then neutralized with 70 ,ul 1 N acetic acid at room temperature and the solid phase was separated from the liquid phase by centrifugation.
The starch was hydrolyzed using a kit for detecting starch (Boehringer, Mannheim) according to the manufacturer's instructions using amyloglucosidase and the released glucose was enzymatically determined.

c) Detection of fattY acids in leaves and seeds Leaf disks (1.1 cm diameter each) or one tobacco seed each were heated for 15 min at 80~C in 1 ml 1 N HCI in methanol under a nitrogen atmosphere after adding 5 ,ug myristic acid as internal standard in a closed glass container. After cooling the mixture down to room temperature 1 ml 0.9 % aqueous NaCI solution and 1 ml n-hexane (p. a.) were added. After extraction of the aqueous phase the organic phase was removed and concentrated with gaseous nitrogen.
The fatty acid methyl esters were separated and quantified by gas chromatography according to Browse et al. (Analytical Biochemistry 152 (1986),141-145).

d) Detection of chlorophvll a and b. antheraxanthine. zeaxanthine and violaxanthine Leaf disks (1.1 cm diameter each) were frozen in liquid nitrogen immediately after the samples were taken. The following steps were taken in a room with dimmed lighting.
The samples were homogenized with 250 ,ul ice-cold 85% acetone in water, the suspension was rinsed for about 30 seconds with nitrogen and then kept on ice for 15 minutes. After centrifugation for 15 minutes at 4~C and 100009 the supernatant was passed through a Millipore-Millex-GV4 sterile hlter device and then rinsed for 30 seconds at 4~C with nitrogen oxide. The samples were then stored at -70~C until time of measurement. The pigments were separated and quantified by high pressure liquid chromatography (HPLC) as described by Zhayer and Bjorkman (J. Chromatogr. 543 (1990), 137-145 ).
As separation column a ZORBAX ODS 5 I~m non-endcapped 250 4,5 mm reversed phase column was used. The pigments were detected by measuring the absorption at 450 nm in a range of 0.04 absorption units (AUFS).

10. Detection of the citrate synthase activity in leaves of tobacco plants For detecting the citrate synthase activity in tobacco plants, leaf samples were homogenized in extraction buffer (50 mM Hepes-KOH pH 7.5; 5 mM MgCI2; 1 mM EDTA; 1 mM EGTA; 10% (vol.lvol.) glycerol; 0.1%
(vol./vol. ) Triton X-100; 4 mU / ml a2-macroglobuline). After centrifugation, the cell-free extracts were used to measure the enzymatic activity.
The activity of the citrate synthase was determined photometrically:
Reaction buffer:
0.1 M Tris-CI pH 8.0 0.1 mM5,5'-dithio-bis(-2-nitrobenzoicacid) 0.3 mM Acetyl-CoA
Reaction volume: 700 ,ul Reaction temperature: 30~C
Protein used: ~ 60 ,ug Start: 7 ~150 mM oxaloacetate Absorption was measured at 412 nm.

The examples serve to illustrate the invention.

Example 1 Construction of the binary plasmid Bin-mHy-lnt For plant transformation, the coding region of the acetyl CoA hydrolase gene from Saccharomyces cerevisiae was amplified using polymerase chain reaction (PCR) starting from genomic Saccharomyces cerevisiae DNA and using the primers AcCoHy1 (5'-GTCAGGATCCATGACMI I ICTMl I IGTTAAAGCAGAGA-3') (Seq ID No. 1 ) and AcCoHy2 (5'-GTCAGGATCCCTAGTCMCTGGTTCCCAGCTGTCGACCTT-3') (Seq ID No. 2).
The sequence of the acetyl CoA hydrolase from Saccharomyces cerevisiae is registered with the GenEmbl data bank under accession no. M31036. The cloning of the acetyl CoA hydrolase gene is described in Lee et al. (Joumal of Biological Chemistry 265 (1990), 7413-7418). The amplified fragment corresponds to the region spanning nucleotides 614 to 2194 of said sequence (accession no. M31036). For this purpose at both the 5' end and the 3' end a BamHI cleavage site was introduced. The 1590 bp PCR fragment which was digested with BamHI was cloned into the BamHI cleavage site of vector pUC9-2 via the additional cleavage sites. Intron PIV2 (189 bp) from plasmid p35S GUS INT (Vancanneyt et al., Mol. Gen. Genet. 220 (1990), 245-250) was amplified via PCR using primers GUS-1 (5'-gtatacgtaagtttctgcttctac-3') (Seq ID No. 3) and GUS-2 (5'-gtacagctgcacatcaacaaattttgg-3') (Seq ID No. 4), digested with SnaBI and Pvull and cloned into the unique BbrPI cleavage site of the acetyl CoA hydrolase gene from Saccharomyces cerevisiae. Correct orientation of the inserted intron (5' end of the intron oriented in direction of the 3' end of the 5' exon) was confirmed by sequence analysis. The plasmid obtained in this manner was designated pUC-Hylnt.
Plasmid pUC-Hylnt was digested with BamHI and the 1779 bp acetyl CoA
hydrolase fragment (with inserted intron) was cloned into the BamHI cleavage site of plasmid pAM. The plasmid obtained in this manner was designated pAM-Hylnt.
Plasmid pAM was produced as follows. The mitochondrial target sequence (111 bp) of the protein of the matrix processing peptidase (MPP) from potato was amplified by PCR using primers Mito-TP1 (5'-GATCGGTACCATGTACAGATGCGCATCGTCT-3') (Seq ID No. 5) and Mito-TP2 (5'-GTACGGATCCCTTGGTTGCMCAGCAGCTGA-3') (Seq ID No. 6).

As matrix for the PCR plasmid pMPP (Braun et al., EMBO J. 11 (1992), 3219-3227) was used. The amplified fragment corresponds to the region spanning nucleotides 299 to 397 of the MPP cDNA (Braun et al., supra; EMBL
accession no.: X66284). For this purpose, an Asp718 cleavage site was introduced at the 5' end and a BamHI cleavage site at the 3' end. The PCR
fragment was digested with Asp718 and BamHI and the resulting 109 bp fragment was then cloned into vector pA7 which had been digested with Asp718 and BamHI (von Schaewen, A. (1989) doctoral thesis, Freie Universitat Berlin).
Plasmid pAM-Hylnt was digested with Asp718 and Xbal and the 1887 bp fragment consisting of the coding region for the targeting peptide of potato "matrix processing peptidase" and the coding region for the acetyl CoA
hydrolase from Saccharomyces cerevisiae (with inserted intron) was isolated and the 5' overhangs of said fragment were blunt-ended with T4-DNA
polymerase. The fragment so produced was cloned into the Smal cleavage site of the binary plasmid pBinAR-Hyg. The coding region of the targeting peptide of potato "matrix processing peptidase" was oriented in direction of the 35S-RNA promoter of the cauliflower mosaic virus. The resulting plasmid Bin-mHy-lnt (see Figure 1) was used for transformation of tobacco (Nicotiana tabacum SNN) and potato (Solanum tuberosum L. cv. Désirée) as described above.
Example 2 Construction of binary plasmid Bin-Hy-lnt The BamHI fragment of plasmid pUC-Hy-lnt (see Example 1) was cloned into the BamHI cleavage site of plasmid pA7 (von Schaewen, A. (1989) doctoral thesis, Freie Universitat Berlin). The 5' end of the coding region of the acetyl CoA hydrolase was oriented in direction of the 35S-RNA promoter. The plasmid so obtained was designated pA7-Hy-lnt. Plasmid pA7-Hy-lnt was digested with Kpnl and Xbal, the 1778 bp fragment consisting of the coding region for acetyl CoA hydrolase from Saccharomyces cerevisiae (with inserted intron) was isolated and then cloned into binary plasmid pBinAR-Hyg (deposition no.: DSM 9505; date of deposition: October 20, 1994) which had been digested with Kpnl and Xbal. Resulting plasmid Bin-Hy-lnt (see Figure 2) was used for the transformation of tobacco (Nicotiana tabacum SNN) and potato (Solanum tuberosum L. cv. Désirée).

Example 3 Analysis of transgenic tobacco plants which express an acetyl CoA
hydrolase from Saccharomyces cerevisiae Whole tobacco plants were regenerated from tobacco plants which had been transformed with plasmid Bin-mHy-lnt (see Example 1), transferred to soil and selected by Western blot analysis for the presence of the acetyl CoA
hydrolase from Saccharomyces cerevisiae in leaves. Several genotypes were identified which clearly express the acetyl CoA hydrolase from Saccharomyces cerevisiae (see Fig. 3). Several of the selected transgenic lines were analyzed for the acetyl CoA hydrolase activity in leaves. In some of the lines a specific acetyl CoA hydrolase activity was measured that was up to three times as high as compared with that of the control plants (e.g., MB-Hy1-39, MB-Hy1-78, MB-Hy1-81; cf. Table 1).

Table I

Plant acetyl CoA hydrolase activity (nmol min~' mg~' protein) Control 1.49 + 1.06 MB-Hy1-39 5.58 + 1.59 MB-Hy1 -78 2.92 + 0.74 MB-Hy1-81 3.25 + 0.79 The enzyme activities indicated above are the average value of at least eight measurements starting from at least three independent plants of the above-mentioned transgenic line.

The above-mentioned genotypes MB-Hy1-39, MB-Hy1-78, MB-Hy1-81 were amplified, and 3 plants of each genotype were transferred to a greenhouse.

It was surprisingly found that the leaves of the transgenic lines MB-Hy1-39, MB-Hy1-78, MB-Hy1-81 contain reduced amounts of fatty acids as compared with control plants (cf. Table ll).

Table ll Typeof Control plant MB-Hy1-39 MB-Hy1-78 MB-Hy1-81 fatty acid[I~mol/g [,umol/g [,umol/g [,umol/g (dried weight)] (dried weight)] (dried weight)] (dried weight)]
16:0 2.63 ~0.35 1.17 0.18 2.00 ~0.34 2.21 iO.29 16:1 0.76 +0.10 0.35 +0.05 0.59 ~0.05 0.65 ~0.07 16:2 0.57 ~0.10 0.23 _0.04 0.31 ~0.02 0.37 ~0.01 18:0 0.26 ~0.06 0.11 ~0.02 0.16 ~0.03 0.18 ~0.02 16:3 1.69 ~0.17 0.66 ~0.13 1.66 ~0.18 0.68 ~0.12 18: 1 0.69 ~0.13 0.15 ~0.00 0.27 ~0.07 1.26 ~0.01 18:2 2.79 ~0.27 1.13 ~0.13 1.44 ~0.13 2.21 ~0.23 18:3 11.43 +1.46 4.36 ~0.60 8.61 ~0.94 10.02 ~0.54 Total: 20.83 _2.64 8.17 +1.15 15.03 i1.78 17.57 +1.29 The values indicated are the average values as well as standard deviations derived from 2 independent measurements each.

It was surprisingly also found that the seeds of the transgenic plants MB-Hy1-39, MB-Hy1-78, MB-Hy1-81 contain identical amounts of fatty acids as compared with control plants (cf. Table lll).

Table lll Typeof Control plant MB-Hy1-39 MB-Hy1-78 MB-Hy1-81 fatty acid [nmollseed] [nmol/seed] [~I"~01~3eedl [",.,ol/32ed]16:0 12.28 i2.86 10.14 +3.90 13.55 i2.63 10.71 +1.20 16:1 0.25 iO.19 0.27 iO.17 0.42 iO.O9 0.27 iO.15 16:2 0.20 iO.17 0.07 iO.11 0.09 iO.12 0.07 iO.08 18:0 0.02 iO.05 0.03 iO.06 0.00 iO.OO 0.01 iO.03 16:3 3.01 ~1.13 2.04 iO.86 3.46 +1.12 2.60 iO.42 18:1 13.08 ~3.49 10.25 i5.02 15.00 i2.99 10.78 ~1.37 18:2 101.49 i20.42 86.29~36.05 111.58 +19.46 90.03 ~8.97 18:3 1.71 iO.28 1.74 iO.60 2.06 iO.35 2.05 iO.37 Total:132.05 i28.59 110.84i46.77 146.16 +10.71 116.51 +12.59 The values indicated are the average values as well as standard deviations derived from 6 independent measurements each.
An analysis of the weight of 200 seeds each showed that there was no significant difference between the seeds of the transgenic plant MB-Hyl-39 and the seeds of control plants (cf. Table IV).

Table IV

Weight of seed [,ug pe- seed]
Control plant 88.0 _6.0 MB-Hy1-39 87.8 +2.5 An analysis of soluble sugars such as glucose, fructose and sucrose surprisingly showed that the leaves of plants of the transgenic lines MB-Hy1-39, MB-Hy1-78, MB-Hy1-81 contained elevated amounts of sucrose, glucose and fructose both at the end of the light phase (cf. Table V) as well as at the end of the dark phase (cf. Table Vl).

Table V

Contents of soluble sugars at the end of the light phase (n=6) Glucose FructoseSucrose ~umol/g(dried ,umol/g(dried ,umol/g(dried wei3ht) wei3ht)wei3ht) Control plant 17.3 _17.5 31.2 _20.0 146.6 _54.5 MB-Hy1-39 79.6 _44.8 105.8 _61.7 294.0 _112.7 MB-Hy1 -78 188.4 _194.2 118.1 +129.3 239.9 _161.1 MB-Hy1 -81 88.6 _59.0 24.3 _39.5 208.8 +148.8 The values indicated are the average values as well as standard deviations derived from 6 independent measurements each.

Table Vl Contents of soluble sugars at the end of the dark ~hase (n=3) Glucose Fructose Sucrose ,umol/g(dried weight) I mol/g(dried weight) ,umol/g(dried weight) Control plant 10.2 _9.2 5.1 _4.4 44.9 _13.9 MB-Hy1-39 30.0 _18.8 28.1 _14.8 150.9 _112.3 MB-Hy1 -78 142.0 i150.3 53.4 _30.2 190.9 +200.8 MB-Hy1-81 8.8 _7.6 12.9 _1.9 16.5 _2.1 The values indicated are the average values as well as standard deviations derived from 3 independent measurements each.

An analysis of the starch surprisingly showed that the leaves of plants of the transgenic lines MB-Hy1-39, MB-Hy1-78, MB-Hy1-81 contained elevated amounts of starch both at the end of the light phase as well as at the end of the dark phase (cf. Table Vll).

Table Vll Content of starch at the end of the light phase and at the end of the dark phase End of the lig lt phase(n=6) End of the dark phase (n=3) mmol/g(dr ed weight) mmol/g(dr ed weight) Control plant2.37 ~0.67 0.97 _0.76 MB-Hy1 -39 4.81 i1.82 2.57 _2.08 MB-Hy1-78 1.79 i1.49 1.77 _2.18 MB-Hy1 -81 2.23 +1.33 0.17 ~0.02 The values indicated are the average values as well as standard deviations derived from 3 and 6 independent measurements each (cf. Table Vll).

When growing the plants MB-Hy1-39, MB-Hy1-78, MB-Hy1-81 in the greenhouse it was furthermore found that the transgenic plants showed a modified phenotype as compared to control plants. Particularly, the transgenic plants exhibited a reduced growth, formation of several shoots as well as a mosaic-like modification in the leaf coloration (see Fig. 4 and 5). For a more detailed analysis of the leaf coloration, the contents of chlorophyll a and b aswell as of the carotinoids zeaxanthine, antheraxanthine and violaxanthine were determined (cf. Tables Vlll and IXa and b).

Table Vlll Content of chlorophyll b Content of chlorophyll a (,umol / g (d-ied weight) (,umol / g (dried weight) Control plant5.42 ~0.46 5.98 _0.54 MB-Hy1-39 2.38 +0.21 2.73 +0.21 MB-Hy1-78 4.23 +0.82 4.81 +0.74 MB-Hy1 -81 5.13 +0.36 6.03 _0.54 The values indicated are the average values as well as standard deviations derived from 3 independent measurements each.

Table IXa Violaxanthine Antheraxanthine Zeaxanthine (~Jmol / g (dried(~mol / g (dried(,umol / g (dried weight) weight) weight) Control plant0.173 _0.042 0.178 _0.049 0.299 _0.114 MB-Hy1 -39 0.215 +0.045 n.d. n.d. n.d. n.d.
MB-Hy1-78 0.314 +0.052 0.051 _0.036 n.d. n.d.
MB-Hy1-81 0.591 +0.205 0.034 +0.004 n.d. n.d.

n.d. not detected (below the limit of detection) Table IXb Luteine Neoxanthine (I~mol/g (dried weight) (,umol/g (dried weight) Control plant1.94 +0.32 0.38 +0.06 MB-Hy1 -39 0.74 +0.11 0.15 _0.03 MB-Hy1-78 1.46 +0.51 0.22 ~0.02 MB-Hy1-81 1.79 +0.40 0.39 ~0.11 The values indicated are the average values as well as standard deviations derived from 3 independent measurements each.

Example 4 Construction of the binary plasmid pTCSAS

Plasmid pTCS, which was obtained by in vivo excision from a ~ ZAP ll cDNA
library from Nicotiana fabacum L., contains a 1747 bp cDNA fragment with the coding region of the citrate synthase gene from tob~cco (~ccession no.
X84226) in the EcoR I cleavage site of vector pBluescript SK.
The BamHI fragment of plasmid pTCS was cloned into the BamHI/Sall cleavage sites of the binary plasmid BinAR. The 3' end of the coding region of the citrate synthase was oriented in direction of the 35S-RNA promoter. The plasmid so obtained was designated pTCSAS.
pTCSAS was used for the transformation of tobacco (Nicotiana tabacum SNN).

Example 5 Analysis of transgenic tobacco plants with reduced citrate synthase activity Regenerated tobacco plants that had been transformed with plasmid pTCSAS
were transferred to soil and selected by measuring the citrate synthase activity in leaves. Several genotypes were identified that clearly showed a reduction in citrate synthase activity (see Table X). Several of the selected transgenic lines were analyzed for the citrate synthase activity in leaves. In some of the lines a specific citrate synthase activity was found that was up to six times as high as compared with that of the control plants (e.g., TCSAS-14, TCSAS-17; TCSAS-26; TCSAS-43; TCSAS-48; cf. Table X).

Table X

Line CS activity [nmol CoA / min mg]
Control plant 69.00 +21.20 TCSAS-14 35.87 +8.62 TCSAS-17 25.21 +7.12 TCSAS-26 11.63 +3.90 TCSAS43 33.90 +8.30 TCSAS48 16.25 +4.80 CS = citrate synthase The enzyme activities indicated above are the average value of at least 18 measurements starting from at least nine independent plants.

The above-mentioned genotypes TCSAS-17 and TCSAS-26 were amplified and 6 plants each were transferred to a greenhouse.
It was surprisingly found that the seeds of the transgenic plants TCSAS-17 and TCSAS-26 contain reduced amounts of fatty acids as compared to control plants (cf. Table Xl).

Table Xl wt t17 t26 [nmol/seed] [nmol/seed~ [nmol/seed]
16:0 15.42 +2.96 7.03 +1.74 9.58 +1.37 18:1 17.78 +4.12 6.88 +2.02 9.24 _2.29 18:2 116.27 +17.6663.25 +12.0980.13 +14.74 18:3 1.74 +0.38 1.23 +0.27 1.44 _0.27 Total: 151.22 _25.1178.39 +16.13100.39 +18.68 The values indicated are the average values as well as standard deviations derived from 10 independent measurements each.

An analysis of the weight of 200 seeds each showed that there was a significant difference between the seeds of the transgenic plants TCSAS-17 and TCSAS-26 and the seeds of control plants (cf. Table Xll).

Table Xll Seed weight [1~9 per seed]
Control plant 90.5 _2.5 TCSAS-17 48.0 1.6 TCSAS-26 60.5+5.5 Example 6 Construction of the plant transformation vector pBin-USP-MTPHylnt For plant transformation, the coding region of the acetyl CoA hYdrolase gene from Saccharomyces cerevisiae was isolated by polymerase chain reaction (PCR) as described in Example 1.
Plasmid pAM-Hylnt was digested witht Asp718 and Hindlll and the 1931 kb fragment consisting of the coding region of the targeting peptide of potato "matrix processing peptidase" and the coding region for the acetyl CoA
hydrolase from Saccharomyces cerevisiae (with inserted intron) was isolated.
The fragment so isolated was cloned into the Asp718/Hindlll cleavage sites of binary plasmid pUSP-Bin19. This vector contains the USP promoter (Fiedler et al., Plant Mol. Biol. 22 (1993), 669-679), particularly the 684 bp Pstl fragment from pP30T, a kanamycin resistance gene, a multiple cloning site and the polyadenylation site of the octopinsynthase gene. The resulting plasmid pBin-USP-MTPHylnt was used for the transformation of rape as described above.

Example 7 Construction of the plant transformation vector pBin-USP/Hyg-TP-ACS

For plant transformation, the coding region of the acetyl CoA svnthetase gene (De Virgilio et al., Yeast 8 (1992), 1043-1051) from Saccharomyces cerevisiae was amplified by polymerase chain reaction (PCR) using primers ACS1 (5'GAT CM GCT TAT GTC GCC CTC TGC CGT ACA ATC -3'; Seq ID No. 7) and ACS2 (5'- GAT CM GCT TTC ATC ATT ACA ACT TGA CCG ATC C-3', Seq ID No. 8) starting from a genomic S. cerevisiae DNA. The sequence of the acetyl CoA synthetase is registered with the GenEMBL data bank under accession no. X66425. Cloning of the acetyl CoA synthetase gene is described in De Virgilio (loc. cit.). The amplified fragment corresponds to the region spanning nucleotides 162 to 2303 of said sequence. For this purpose, a Hindlll cleavage site was introduced at the 5' end and at the 3' end. The 2151 bp Hindlll fragment was cloned into the Hindlll cleavage site of vector pSK-TP via the additional cleavage sites. Said plasmid contains a DNA
sequence which encodes the plastidic transit peptide of the ferredoxin:NADP+
oxireductase from spinach. Plasmid pSK-TP-ACS was digested with Asp718, the cleavage sites were blunt-ended using T4-DNA polymerase and then digested with Xbal. The thus obtained 2380 kb fragment consisting of the coding region of the targeting peptide of spinach ferre-doxin:NADP+ oxireductase and the coding region for the acetyl CoA
synthetase from S. cerevisiae was cloned into the Smal/Xbal cleavage sites of the binary plasmid pBin-USP-Hyg. Said vector contains the USP promoter (684 Pstl fragment from pP30T (Fiedler et al., loc. cit.). The resulting plasmidpBin-USP/Hyg-TP-ACS was used as described above for the transformation of rape plants which express an acetyl CoA hydrolase from yeast (see Example 4).

Example 8 Construction of the plant transformation vector pBin-USP/Hyg-TP-ACLY

For plant transformation, the coding region of the ATP:citrate Iyase gene from Raffus norvegicus was amplified by polymerase chain reaction (PCR) starting from Raffus norvegicus cDNA and using primers ACLY1 (5'- ACT GM GCC
TAT GTC AGC CM GGC MT TTC AGA GCA-3', Seq ID No. 9) and ACLY2 (5'-ACT GM GCC m ACA TGC TCA TGT GTT CCG GGA GM C -3', Seq ID No. 10). The sequence of the ATP:citrate Iyase is registered with the GenEMBL data bank under the accession no. J05210. Cloning of the ATP:citrate Iyase gene from Raffus norvegicus is described in Elshourbagy et al. (J. Biol. Chem. 265 (1990), 1430-1435). The amplified fragment corresponds to the region spanning nucleotides 73 to 3375 of said sequence.
For this purpose a Hindlll cleavage site was added at the 5' end and at the 3' end. The 3312 bp Hindlll fragment was cloned into the Hindlll cleavage site of vector pSK-TP via the additional cleavage sites. Plasmid pSK-TP-ACLY was digested with Asp718, blunt-ended using T4-DNA polymerase and was then digested with Xbal. The thus isolated 3494 kb fragment consisting of the coding region of the targeting peptide of spinach ferredoxin: NADP+
oxireductase and the coding region for the ATP:citrate Iyase from Raffus norvegicus was cloned into the Smal/Xbal cleavage sites of the binary plasmid pBin-USP-Hyg. The resulting plasmid pBin-USP/Hyg-TP-ACLY was used for the transformation of rape as described above.

Example 9 Construction of the plant transformation vector pBin-USP-MTPCS

For plant transformation, the coding region of the citrate sYnthase gene from E. coli was amplified by polymerase chain reaction (PCR) starting from genomic E. coli DNA and using primers CS1 (5'- A CTG GGA TCC ATG GCT
GAT ACA MA GCA AAA CTC ACC C -3', Seq ID No. 11) and CS2 (5'- A
CTG GGA TTC TTA ACG CTT GAT ATC GCT TTT AAA G -3', Seq ID No.
12). Cloning and the sequence of the citrate synthase gene is desuibed Ner et al., Biochemistry 22 (1983), 5243-5249. The amplified fragment corresponds to the coding region spanning nucleotides 1 to 1284 of said sequence. For this purpose, a BamHI cleavage site was introduced at the 5' end and at the 3' end. The 1294 BamHI was cloned into the BamHI cleavage site of vector pAM. Plasmid pAM-CS was digested with Asp718 and Hindlll and blunt-ended with T4-DNA polymerase and the 1393 kb fragment consisting of the coding region of the targeting peptide of potato "matrix processing peptidase" and the coding region for the citrate synthase from E.
coli was isolated. The thus isolated fragment was cloned into the Smal cleavage sites of the binary plasmid pUSP-Bin19 (see Example 6). The resulting plasmid pBin-USP-MTPCS was used as described above for the transformation of tobacco plants as well as of rape plants.

Example 10 Transgenic plants with a mitochondrial acetyl CoA hydrolase and a plastidic acetyl CoA synthetase Plants which co-express an acetyl CoA hydrolase from yeast with mito-chondrial targeting and an acetyl CoA synthetase from yeast with plastidic targeting were regenerated and grown in a greenhouse. The seeds of these plants were examined as described above for their total fatty acid content. An increase by about 5% based on the total fatty acid content (per seed) was found as compared to non-transformed plants.

~ .. .. .. . ..... ... . . . . . . . . .. .. .

Table Xlll Line Fatty acid content ~ Fatty acid content [nmol/seed] [+/-]
Control 133 11 t1 144 12 t2 147 28 t3 145 17 t4 143 18 t5 146 12 ~ .. , .. -- . . . . . . . . .

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:
(A) NAME: Max-Planck-Gesellschaft zur Forderung der Wissenschaften e.V.
(B) STREET: none (C) CITY: Berlin (E) COUNTRY: Germany (ii) TITLE OF THE INVENTION: Transgenic plant cells and plants with modified acetyl CoA production (iii) NUMBER OF SEQUENCES: 12 (iv) COMPUTER-READABLE FORM:
(A) MEDIUM TYPE: floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.30 (EPA) (2) INFORMATION FOR SEQ ID NO: 1:

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Claims (23)

1. Transgenic plant cell with a modified acetyl CoA metabolism which due to the expression of a foreign DNA sequence encoding a protein with acetyl CoA hydrolase activity exhibits an elevated acetyl CoA hydrolase activity as compared to wild-type cells.

2. The transgenic plant cell according to claim 1, wherein the acetyl CoA
hydrolase activity in the mitochondria is elevated.

3. The transgenic plant cell according to claim 2, which furthermore exhibits an elevated acetyl CoA synthetase activity in the cytosol.

4. The transgenic plant cell according to claim 1, wherein the acetyl CoA
hydrolase activity in the cytosol is elevated.

5. The transgenic plant cell according to claim 2 or 4, which furthermore exhibits an elevated acetyl CoA synthetase activity in the plastids.

6. The transgenic plant cell according to any one of claims 1 to 5, which furthermore exhibits a reduced citrate synthase activity in the mitochondria.

7. The transgenic plant cell according to any one of claims 1 to 5, which furthermore exhibits an elevated citrate synthase activity in the mitochondria or in the cytosol.

8. The transgenic plant cell according to any one of claims 1 to 7, which furthermore exhibits a reduced ATP citrate lyase activity in the cytosol.

9. The transgenic plant cell according to any one of claims 1 to 7, which furthermore exhibits an elevated ATP citrate lyase activity in the cytosol.

10. The transgenic plant cell according to any one of claims 1 to 9, wherein the acetyl CoA hydrolase is an unregulated or deregulated enzyme.

11. The transgenic plant cell according to claim 10, wherein the acetyl CoA
hydrolase originates from fungal cells.

12. The transgenic plant cell according to claim 11, wherein the acetyl CoA
hydrolase originates from Saccharomyces cerevisiae.

13. The transgenic plant cell according to claim 10, wherein the acetyl CoA
hydrolase originates from a prokaryotic organism.

14. The transgenic plant cell according to any one of claims 3 or 5 to 13, wherein the acetyl CoA synthetase is an unregulated or deregulated enzyme.

15. The transgenic plant cell according to claim 14, wherein the acetyl CoA
synthetase originates from a fungal, bacterial or animal organism.

16. The transgenic plant cell according to claim 15, wherein the acetyl CoA
synthetase originates from Saccharomyces cerevisiae.

17. Plant containing plant cells according to any one of claims 1 to 16.

18. The plant according to claim 17, which exhibits at least one of the following features:
(a) a reduced or elevated content of fatty acids in the leaf tissue or in the seed tissue as compared to wild-type plants;
(b) an elevated content of soluble sugars in the leaf tissue as compared to wild-type plants;
(c) an elevated content of starch in the leaf tissues as compared to wild-type plants;
(d) reduced growth;
(e) formation of two or more shoots;
(f) modified leaf coloration.

19. The plant according to claim 17 or 18, which is an oil-storing plant.

20. Propagation material of a plant according to any one of claims 17 to 19, which contains transgenic plant cells according to any one of claims 1 to 16.

21. The propagation material according to claim 20, which is a fruit, a seed or a tuber.

22. Use of DNA sequences which encode a protein with the enzymatic activity of an acetyl CoA hydrolase for expression in plant cells in order to increase the activity of the acetyl CoA hydrolase in plant cells.

23. The use according to claim 22, wherein the acetyl CoA hydrolase activity in the cytosol is elevated.