WO1998006831A1

WO1998006831A1 - Transgenic plant cells and plants with modified acetyl-coa formation

Info

Publication number: WO1998006831A1
Application number: PCT/EP1997/004311
Authority: WO
Inventors: Lothar Willmitzer; Bernd Müller-Röber; Ursula La Cognata; Michael BÄUERLEIN
Original assignee: MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
Priority date: 1996-08-08
Filing date: 1997-08-07
Publication date: 1998-02-19
Also published as: EP0918849A1; CA2263186A1; AU739905B2; AU4203997A

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

Transgenic plant cells and plants with altered

Acetyl-CoA formation

The present invention relates to transgenic plant cells and plants with a modified acetyl-CoA metabolism compared to non-transformed plants and with a changed ability to form and utilize acetyl-CoA (acetyl-coenzyme A). The change in the ability to produce and use acetyl-CoA is accomplished by the introduction and expression in plant cells of a DNA sequence encoding an acetyl-CoA hydrolase, preferably a deregulated or unregulated acetyl-CoA hydrolase. The invention also relates to the use of DNA sequences which encode an acetyl-CoA hydrolase to increase the acetyl-CoA hydrolase activity in plant cells, in particular for the production of transgenic plant cells and plants, with modified ability to form and utilize acetyl -CoA.

Due to the continuously increasing demand for food, which results from the constantly growing world population, one of the tasks of biotechnological research is to strive to increase the yield of crops. One way to achieve this is by genetically modifying the metabolism of plants. The goals are, for example, the primary processes of photosynthesis, which lead to CO2 fixation, the transport processes, which are involved in the distribution of the photosassiates within the plant, as well as metabolic pathways, which are used to synthesize storage substances, e.g. starch, proteins, Fats, oils, rubber substances, or of secondary metabolites such as flavonoids, steroids, isoprenoids (eg flavorings), pigments or polyketides (antibiotics), or of plant pathogen defense agents. While many applications are concerned with the steps that either lead to the formation of photoassimilates in leaves (cf. also EP-A 0 466 995) or with the Formation of polymers such as starch or fructans in storage organs of transgenic plants (eg WO 94/04692), there are no promising approaches to date which describe which modifications have to be introduced in the primary metabolic pathways in order to change the ability to form and utilize acetyl To achieve CoA. A change in the ability to form and utilize acetyl-CoA is important, for example, for all those processes in the plant for which larger amounts of acetyl-CoA are required. This applies, for example, to many biochemical processes in plant cells, in which acetyl-CoA is involved as a substrate or degradation product. A change in the acetyl-CoA formation rate is particularly important for the formation of starch, proteins, fats, oils, gums, or of secondary metabolites such as flavonoids, steroids, isoprenoids (flavorings, pigments), or polyketides (antibiotics). Since plants would be suitable for the production of various of the above-mentioned substances on a large scale due to different properties, there is a need for plants in which the formation and distribution of acetyl-CoA in the cells is changed in such a way that the formation of the above-described substances is influenced becomes.

The present invention is therefore based on the object of making available plant cells and plants with a modified ability to form and utilize acetyl-CoA, and also processes for their production.

The solution to this problem is provided by the provision of the embodiments described in the patent claims.

Thus, the present invention relates to transgenic plant cells with an altered acetyl-CoA metabolism which, due to the expression of a foreign DNA sequence which encodes a protein with acetyl-CoA hydrolase activity, one compared to wild-type, ie non-transformed, cells have increased acetyl-CoA hydrolase activity. The expression of such a DNA sequence leads to an increase in the intracellular acetyl-CoA hydrolase activity in the transgenic plant cells. Acteyl-CoA hydrolases are enzymes that catalyze the following reaction:

Acetyl-CoA < ^■ - "> acetate + HSCoA.

It has surprisingly been found that it is possible to increase the acetyl-CoA hydrolase activity in various compartments of plant cells and thus to influence the intracellular distribution of metabolites. This result is surprising in that acetyl-CoA is one of the central metabolites in plant and animal cells, the concentration of which is strictly regulated (Randall and Miernyk, Method in Plant Biochemistry Vol 3 [ISBN 0-12-461013-7] ). An intervention in the intracellular acetyl-CoA distribution should therefore have a drastic impact on the viability of the cells. For example, there are indications that the expression of acetyl-CoA hydrolase from yeast in E. coli has a lethal effect. In contrast, the present invention is based on the fact that an increase in acetyl-CoA hydrolase activity in plant cells is indeed possible and leads to advantageous properties of the plant cells. For example, it was found that the increase in acetyl-CoA hydrolase activity in the mitochondria in the leaves of transgenic plants leads to an increase in the content of soluble sugars, such as, for example, glucose, fructose and sucrose, and in starch, and to a simultaneous reduction in the content of fatty acids. This means that the increase in mitochondrial acetyl-CoA hydrolase activity enables a change in the partitioning of photoassimilates in the cells. The acetyl-CoA hydrolase activity in the cells according to the invention is preferably increased by at least 50% and particularly preferably by 100% compared to non-transformed cells. Special Another advantage is an increase in enzyme activity by more than 150% compared to non-transformed cells. The increase in acetyl-CoA hydrolase activity leads to an increased concentration of acetate. In contrast to acetyl-CoA, acetate can penetrate unregulated cellular membranes. It is therefore available in other cellular compartments in higher concentration as a substrate for the acetyl-CoA synthesis catalyzed by the acetyl-CoA synthetase. This means that it is possible, by increasing the acetyl-CoA hydrolase activity in one compartment, to change the intracellular distribution of acetyl-CoA and thus to influence the flow of metabolites in different biosynthetic pathways.

In a preferred embodiment, the present invention relates to transgenic plant cells in which the acetyl-CoA hydrolase activity is increased in the mitochondria. In plant cells, acetyl-CoA is biosynthesized in the mitochondria through the pyrurate-dehydrogenase multienzy complex-catalyzed conversion of pyrurate. The increase in acetyl-CoA hydrolase activity in the mitochondria leads to an increased concentration of acetate, which through cellular membranes in other compartments, e.g. can diffuse into the cytosol. Here again it can be used for the synthesis of acetyl-CoA, e.g. can be used by the acetyl-CoA synthetase.

In a further preferred embodiment, the transgenic plant cells according to the invention therefore show an increased activity of the acetyl-CoA synthetase in the cytosol. This enzyme catalyzes the following reaction

Acetate + HSCoA -r AcetylCoA + AMP + PPι

The acetyl-CoA that is increasingly formed in the cytosol can be used, for example, for an increased synthesis of isoprenoids by means of valonic acid and isopentenyl pyrophoshate can be used (Bach, Lipids 30 (1995), 191-202).

In another preferred embodiment, the acetyl-CoA hydrolase activity is increased in the cytosol of the transgenic plant cells. This in turn leads to an increased acetate concentration. For example, this can lead to more acetate being available in the plastids and being converted into acetyl-CoA in these. This would increase acetyl-CoA as a substrate e.g. for fatty acid biosynthesis or isoprenoid synthesis.

In a particularly preferred embodiment, the transgenic plant cells according to the invention with an increased acetyl-CoA hydrolase activity in the mitochondria or the cytosol additionally show an increased activity of the acetyl-CoA synthetase in the plastids. This can increase the shift of acetate into the plastids and its conversion into acetyl-CoA. This is then available, for example, to an increased extent for fatty acid biosynthesis.

In principle, it is also possible to reduce the acetyl-CoA synthetase activity in the cytosol.

In a further preferred embodiment, the plant cells described above have a reduced activity of citrate synthase in the mitochondria. This enzyme catalyzes the following reaction:

Acetyl-CoA + oxaloacetate -> citrate + HSCoA

By reducing the activity of this enzyme, which uses acetyl-CoA as a substrate for citrate synthesis, more acetyl-CoA is available for the reaction catalyzed by the acetyl-CoA hydrolase, which leads to an increased formation of acetate. Another preferred embodiment of the present invention provides that the activity of citrate synthase is increased in the plant cells according to the invention described above in the mitochondria or the cytosol.

Such an increase in the activity of citrate synthase can lead to a change in the flow of metabolites to acetyl-CoA in a specific subcellular compartment, and can in particular cause an increase in the biosynthesis of fatty acids or lipids.

In a further preferred embodiment of the present invention, the plant cells according to the invention described above also have a reduced activity of the ATP citrate lyase in the cytosol. This enzyme catalyzes the following reaction:

Citrate + HSCoA + ATP <-> acetyl-CoA + AMP + PPi + oxaloacetate.

Such a reduction can lead to an increase in the metabolic flow from citrate to acetyl-CoA, which can cause a build-up of metabolites in the citrate cycle. It would therefore be possible to increase the formation of acetate via the endogenous acetyl-CoA hydrolase, as a result of which the formation of lipids and / or terpenoids can be increased.

Another embodiment of the present invention provides that the plant cells have an increased ATP citrate lyase activity in the cytosol.

Such an increase can result in an increased formation of cytosolic acetyl-CoAs, which can lead to an increased synthesis of isopentenyl pyrophosphate (IPP) and thus to an increased formation of terpenoids.

The transgenic plant cells described above have, due to the modified enzyme activities described, a modified ability to form and utilize acetyl-CoA compared to wild-type cells. This can be done, for example, through the Determination of the changed 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 have altered amounts of isoprenoids, steroids, pigments, isoprenoids, flavonoids, hormones, fats, oils, proteins, rubber substances, polyketides or substances which are involved in the defense against plants, or their soluble content Sugars, such as glucose, fructose and sucrose, as well as changes in starch. Such cells can in turn be advantageous starting materials for further uses. For example, these cells can be used for the heterologous expression of further genes with the aim of intensifying the synthesis of economically relevant substances. For example, DNA sequences can be introduced which encode the enzymes for the synthesis of polyhydroxyalkanoic acids (eg PHB and PHA). In this way, the increasingly formed acetyl-CoA can be used in plants for the synthesis of such acids, which are of great economic importance. Other examples of economically interesting substances are polyketides, flavorings, rubber, alkaloids, isoprenoids etc.

Of particular importance is the possibility, by expression of an acetyl-CoA hydrolase in oil-storing tissues of a plant, such as the endosperm or the cotyledons of seeds or in other oil-storing organs, the flow of the photoassimilates delivered in the seeds or organs in the direction of Formation of sugars, starches, fats, pigments, isoprenoids, polyketides, steroids, flavonoids, gums, substances that are involved in plant pathogen defense, proteins, and polymers such as polyhydroxyalkanoic acids (see e.g. Poirier et al., Bio / Technology 13 ( 1995), 142-150). General advantages of the cells according to the invention are the possibility of influencing the partitioning of metabolic metabolites, in particular acetyl-CoA, on the content of metabolic end products such as starch and fats, the content and the composition. Settlement of secondary metabolites, the energy balance and on 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 tissue, e.g. the endosperm or cotyledons of seeds or other oil-storing organs. Such cells preferably have a fat content which is at least 3%, preferably at least 5% and particularly preferably at least 7% higher than that of corresponding cells from non-transformed plants.

The acetyl-CoA hydrolase activity in the cells according to the invention is preferably increased by introducing and expressing DNA sequences which code for an acetyl-CoA hydrolase. These DNA sequences, which encode a protein with the enzymatic activity of an acetyl-CoA hydrolase, can be both prokaryotic, in particular bacterial, and eukaryotic DNA sequences, i.e. DNA sequences from plants, algae, fungi or animal organisms or sequences which encode acetyl-CoA hydrolases from such organisms.

In a preferred embodiment of the invention, the DNA sequences which encode an acetyl-CoA hydrolase are sequences which encode enzymes which are deregulated or unregulated in comparison to acetyl-CoA hydrolases normally found in plant cells. Deregulated means that these enzymes are not regulated in the same way as the acetyl-CoA hydrolase enzymes normally formed in unmodified plant cells. In particular, these enzymes are subject to other regulatory mechanisms, ie they are not inhibited to the same extent by the inhibitors present in the plant cells or are allosterically regulated by metabolites. Deregulated preferably means that the enzymes have a higher activity than endogenously expressed acetyl-CoA hydrolases in plant cells. In the context of this invention, unregulated means that the enzymes in plant cells are not subject to any regulation. These enzymes encoded by the sequences can be both known enzymes occurring in nature which have different regulation by various substances and also enzymes which, by mutagenesis of DNA sequences, the known enzymes from bacteria, algae, fungi , Encode animals or plants.

In a particularly preferred embodiment of the present invention, the DNA sequences used encode proteins with the enzymatic activity of an acetyl-CoA hydrolase from fungi, in particular from fungi of the genus Saccharomyces. DNA sequences which encode an acetyl-CoA hydrolase from Saccharomyces cerevisiae are preferably used. Such sequences are known and described (see Lee et al., Journal of Biological Chemistry 265 (1990), 7413-7418 (accession number M31036)). In order to ensure the localization of acetyl-CoA hydrolase in mitochondria of the plant cells, DNA sequences which code for mitochondrial targeting sequences must be fused with 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 DNA sequence mentioned from Saccharomyces cerevisiae, other DNA sequences are also known which code for proteins with the enzymatic activity of an acetyl-CoA hydrolase, for example from Neurospora crassa (see EMBL accession number M31521; Marathe et al., Molecular and Cellular Biology 10 (1990), 2638-2644) and, because of their properties, can also be used to produce the plant cells according to the invention. Care must be taken to ensure that the protein is formed in mitochondria or in the cytosol of the plant cell. Techniques for modifying such DNA sequences to localize them to ensure the synthesized enzymes in mitochondria and in the cytosol of plant cells are known to the person skilled in the art. In the event that the acetyl-CoA hydrolases contain sequences which are necessary for secretion or for a specific subcellular localization, for example for localization in the extracellular space or the vacuole, the corresponding DNA sequences must be deleted. Furthermore, DNA sequences which code for an acetyl-CoA hydrolase can be isolated from any organism with the aid of the already known DNA sequences. Methods for the isolation and identification of such DNA sequences are known to the person skilled in the art, for example hybridization with known sequences or by polymerase chain reaction using primers which are derived from known sequences.

The enzymes encoded by the identified DNA sequences are then examined for their enzyme activity and regulation.

By introducing mutations and modifications according to techniques known to those skilled in the art, the regulatory properties of the proteins encoded by the DNA sequences can be changed further in order to obtain de-regulated or unregulated enzymes compared to acetyl-CoA hydrolases naturally occurring in plants.

The increase in acetyl-CoA synthase, citrate synthase or ATP citrate lyase activity in the plant cells according to the invention is preferably achieved by the introduction and expression of DNA sequences which code for such enzymes. These sequences can be sequences which produce such enzymes from prokaryotic, in particular bacterial, or from eukaryotic organisms, e.g. Encode plants, algae, fungi or animals.

In a preferred embodiment, such enzymes are deregulated or unregulated enzymes, as explained above in connection with the acetyl-CoA hydrolase. These enzymes encoded by the sequences can be both known enzymes occurring in nature, which have a different regulation by different substances, as well as enzymes, which by mutagenesis of DNA sequences, the known enzymes from bacteria, algae, Encode mushrooms, animals or plants.

DNA sequences encoding acetyl-CoA synthases from various organisms have been described. In animal organisms e.g. those known from Macropusengenii, Mensch, Caenorhabditiε elegans and Drosophila melanogaster (see e.g. GenEMBL database accession numbers 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, in particular from those of the genus Saccharomyces, and particularly preferably from Saccharomyces cerevisiae. Such sequences are accessible, for example, under the GenEMBL database access numbers Z47725, M94729, L09598, X56211 for Saccharomyces cerivisiae, in particular under X76891. It is also possible to use DNA sequences which encode bacterial acetyl-CoA synthetases. Such are e.g. accessible under the access numbers M97217, M87509 or M63968.

DNA sequences encoding a citrate synthase are known from various organisms. Sequences which encode vegetable citrate synthases are known, for example, for Arabidopsis thaliana (GenEMBL database access number X17528; Unger et al., Plant Mol. Biol. 13 (1989), 411-418), and for tobacco, potato and sugar beet (see WO 95/24487). Furthermore, sequences are known which code animal citrate synthases, for example from pigs (accession number M21197, Evans et al., Biochemistry 27 (1988), 4680-4686). Sequences are preferably used which contain a citrate synthase with the biological Properties of a citrate synthase from bacteria, especially E. coli, or fungi, in particular Saccharomyces cerevisiae, code. Various sequences encoding bacterial citrate synthases are available, for example, under the GenEMBL database accession numbers: M33037, Z70021, M74818, Z70017, Z70009, Z70016, L38987, Z70014, Z70022, Z70019, Z70018, Z70020, Z70012, Z70010, Z70010 Z70013, Z70015, M36338, L33409, X66112, X60513, Z73101, M29728, M17149, L41815, Z34516, M73535, L14780, X55282, L75931 and D90117. A preferred sequence used is that in Ner et al. (Biochemistry 22 (1983), 5243-5249), which published a citrate synthase from E. coli coded. Sequences that citrate synthases from E. encoding coli are available, for example, under accession numbers M28987 and M28988 (see also Wilde et al., J. Gen. Microbiol. 132 (1986), 3239-3251). Sequences encoding citrate synthases from fungi are available under accession numbers D63376 and D69731, those from S. cerevisiae in particular under the access numbers Z11113, Z48951, Z71255, M54982, X88846 and X00782. The latter is preferred.

DNA sequences encoding an ATP citrate lyase are known, for example, from Rat (Elshourbagy et al., J. Biol. Chem. 265 (1990), 1430-1435), human (Elshourbagy et al., Eur. J. Biochem 204 (1992), 491-499), C. elegans (Wilson et al., Nature 368 (1994), 32-38) and Arabidopsis thaliana (EMBL accession numbers T13771, Z18045, Z25661 and Z26232).

What has already been said above in connection with the acetyl-CoA hydrolase applies to the localization of the respective enzyme in the desired compartment of the plant cell.

The activity of citrate synthase or ATP-citrate lyase in the cells according to the invention can be reduced by methods known to the person skilled in the art, for example by express sion of an antisense RNA, a specific ribozyme or by means of a cosuppression effect.

In order to ensure the expression of the DNA sequences which code for the enzymes described above in plant cells, they can in principle be placed under the control of any promoter which is functional in plant cells. The expression of the 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, but preferably takes place in those tissues in which an altered ability to form and utilize acetyl-CoA is advantageous either for the growth of the plant or for the formation of ingredients within the plant. Promoters which ensure specific expression in a specific tissue, at a specific time of development of the plant or in a specific organ of the plant therefore appear to be particularly suitable. The DNA sequences are preferably under the control of promoters which ensure seed-specific expression. In the case of starch-storing plants, e.g. In maize, wheat, barley or other cereals, this changes the ability of acetyl-CoA to form and utilize in the seeds, and there is a change in the synthesis of seeds.

In a preferred embodiment, promoters are used for increasing the fatty acid biosynthesis as a result of an increased acetyl-CoA content in seeds of oil-forming plants such as oilseed rape, soybean, sunflower and oil palms, which are specifically active in the endosper or in the cotyledons of seeds which form, such as the Phaseolin promoter from Phaseolus is vulgar, the USP promoter from Vicia faba or the HMG promoter from wheat.

According to the invention, it is also advantageous to use promoters for expressing the DNA sequences which are stored in Organs such as tubers or roots are active, for example in the storage root of the sugar beet or in the tuber of the potato. In this case, for example, the expression of the DNA sequences which encode an acetyl-CoA hydrolase leads to a redirection of biosynthetic pathways in the sense of the formation of more sugar or starch and a changed formation and utilization of acetyl-CoA in the direction of fatty acid biosynthesis.

Furthermore, the expression of the DNA sequences can take place under the control of promoters which are activated specifically at the time of flowering induction, or during flowering, or which are active in tissues which are necessary for flowering induction. Promoters can also be used which are activated at a time controlled only by external influences, e.g. by light, temperature, chemical substances (ε. for example WO 93/07279). For increasing the export rate of photoassimilates from the sheet, e.g. Promoters of interest that have a cell-specific expression. Such promoters are known (e.g. the promoter of the rolC gene from Agrobacterium rhizogenes).

The DNA sequences which code for the enzymes described above are preferably linked, in addition to a promoter, to DNA sequences which ensure a further increase in transcription, for example so-called enhancer elements, or to DNA sequences which are in the transcribed region and which ensure a more efficient translation of the synthesized RNA into the corresponding protein. Such regions can be obtained from viral genes or suitable plant genes or can be produced synthetically. They can be homologous or heterologous to the promoter used. Advantageously, the coding DNA sequences are also linked to 3 'untranslated DNA sequences which ensure the termination of the transcription and the polyadenylation of the transcript. Such sequences are known and described, for example that of the octopine synthase gene from Agrobacterium tumefaciens. These sequences are interchangeable.

The DNA sequences which are introduced and expressed in plant cells according to the invention are preferably stably integrated into the genome in the plant cells according to the invention. In addition to the modified enzyme activities as described above, the transgenic plant cells according to the invention can also be distinguished from non-transformed plant cells in that they have a foreign DNA stably integrated in the genome, the expression of which changes the acetyl-CoA hydrolase activity and, if appropriate, a further one of the above Enym activities described causes. In this context, foreign DNA means that the DNA is either heterologous with respect to the transformed plant species, or the DNA, if it is homologous to it, is located at a location in the genome where it is not in non-transformed cells occurs. This means that the DNA is in a genomic environment in which it does not occur naturally. Furthermore, the foreign DNA usually has the characteristic that it is recombinant, i.e. consists of several components that do not occur in nature in this combination.

The transgenic plant cells according to the invention can in principle be cells of any plant species. Of interest are both cells of monocotyledonous as well as dicotyledonous plant species, in particular cells that store starch or agricultural crops, such as e.g. Rye, oats, barley, wheat, potatoes, corn, rice, peas, sugar beets, tobacco, cotton, wine, tomatoes etc. or cells of ornamental plants.

In a preferred embodiment, these are plant cells of oil-storing useful plants, such as, for example, rapeseed, sunflower, oil palm or soybean. Rapeseed is particularly preferred. The present invention furthermore relates to transgenic plants which contain transgenic plant cells according to the invention. Such plants can be produced, for example, by regeneration from plant cells according to the invention by methods known to the person skilled in the art.

Plants containing cells according to the invention preferably have at least one of the following features:

(a) a reduced or increased content of fatty acids in the leaf tissue or in the seed tissue compared to wild-type plants;

(b) an increased content of soluble sugars in the leaf tissue compared to wild-type plants;

(c) an increased starch content in leaf tissue compared to wild-type plants;

(d) reduced growth;

(e) formation of two or more rungs;

(f) change in leaf color.

The invention furthermore relates to propagation material of plants according to the invention which contains cells according to the invention. These include, for example, cuttings, fruit seeds, rhizomes, tubers, seedlings etc.

The present invention also relates to recombinant DNA molecules which contain the following elements:

(a) a promoter functional in plant cells, and

(b) a DNA sequence which encodes a protein with the enzymatic activity of an acetyl-CoA hydrolase and which is linked to the promoter in such a way that it can be transcribed into a translatable RNA in plant cells.

The transfer of the DNA molecules which contain DNA sequences which encode one of the enzymes described above takes place according to methods known to the person skilled in the art, preferably using plasmids, in particular such plasmids which ensure stable integration of the DNA molecule into the genome of transformed plant cells, for example binary plasmids or Ti plasmids from the Agrojacteriujn tume facienε system. In addition to the A ro-acte-riu-n system, other systems for introducing DNA molecules into plant cells are also possible, such as 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 onocotyledonous and dicotyledonous plants are described in the literature and are known to the person skilled in the art.

Finally, the present invention relates to the 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 acetyl-CoA hydrolase activity in plant cells. In particular, the invention relates to the use of such DNA sequences for the production of transgenic plant cells which, compared to non-transformed plant cells, have an altered ability to form sugars, starches, fats, pigments, isoprenoids, polyketides, steroids, flavonoids, rubber substances, substances which are involved in plant pathogen defense, have proteins and / or polymers such as polyhydroxyalkanoic acids. The acetyl-CoA hydrolase activity is preferably increased in the mitochondria or in the cytosol of the plant cells.

1 shows a schematic illustration of the 14.39 kb plasmid Bin-mHy-Int. The plasmid contains the following fragments:

A = fragment A (528 bp) contains the EcoRI-Aεp718 fragment of the promoter region of the 35S promoter of the

"Cauliflower Mosaic Virus" (nucleotides 6909 to 7437) (Frank et al., 1980, Cell 21 (285-294)). B = fragment b (109 bp) comprises a DNA fragment with the coding region of the mitochondrial target sequence of the Proteins of the potato matrix processing peptidase (MPP) (Braun et al., EMBO J. 11 (1992), 3219-3227 (accession number X66284)).

C = fragment C (189 bp) comprises a DNA fragment of the intron PIV2 from the plasmid p35S GUS INT (Vancanneyt et al., Mol. Gen. Genet. 220 (1990), 245-250).

D '= fragment D ¹ (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 number 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 265 (1990), 7413-7418), nucleotides 785 to 2194 (accession number 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).

2 shows a schematic illustration of the 14.28 kb plasmid Bin-Hy-Int. 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 osaic virus" (nucleotides 6909 to 7437) (Frank et al., Cell 21 (1980), 285-294).

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 number 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 number 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) .

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

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

5 shows leaves of the transgenic MB-Hyl lines 39

Fig. 6 shows leaves of a control plant

Fig. 7 shows a plant of a transgenic MB-Hyl line with flowers

8 shows a control plant with flowers

FIG. 9 shows a schematic illustration of the 14, 25 kb Plas ids 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 number X84226). C = fragment C (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) .

Methods

1. Cloning procedure

For cloning in E. coli, the vectors pUC9-2, pA7 (from Schaewen, A. (1989) dissertation, Freie Universität Berlin) and pAM (see Example 1) were used. For the plant transformation, the gene constructions were cloned into the binary vector pBinAR-Hyg (deposit number: DSM 9505; filing date: October 20, 1994).

2. Bacterial strains

For the pUC vectors and for the pBinARHyg constructs, the E. coli strain DH5α (Bethesda Research Laboratories, Gaithersburgh, USA) was used.

The transformation of the plasmids into the potato plants was carried out with the help of the Agrobacterium tume faciens strain C58C1 pGV2260 (Deblaere et al., Nucl. Acids Res. 13 (1985), 4777-4788).

3. Transformation of Agrobacterium tumefaciens

The DNA was transferred by direct transformation using the method of Höfgen and Willmitzer (Nucleic Acids Res. 16 (1988), 9877). The plasmid DNA of transformed agrobacteria was determined by the method of Birnboim and Doly (Nucleic Acids Res. 7 (1979), 1513-1523) isolated and analyzed by electrophoresis after suitable restriction cleavage.

4. Transformation of tobacco or rapeseed

The tobacco was transformed according to the method described in Rosahl et al. (EMBO J. 6 (1987), 1155-1159).

The transformation of rapeseed (Brassica napus) was carried out analogously to the method described in Gene and Transfer in Plants (Springer Verlag Heidelberg (1995), pp. 30-38).

5. Transformation of potatoes

Ten small leaves of a potato sterile culture (Solanum tuberosum L. cv. De εiree) wounded with the scalpel were placed in 10 ml of MS medium (Murashige and Skoog, Physiol. Plant 15 (1962), 473) with 2% sucrose, which contained 50 μl of an Agrobacterium tume aciens culture grown under selection overnight. After gently shaking for 3-5 minutes, another incubation was carried out for 2 days in the dark. The leaves were then used for callus induction on MS medium with 1.6% glucose, 5 mg / 1 naphthylacetic acid, 0.2 mg / 1 benzylaminopurine, 250 mg / 1 claforan, 3 mg / 1 hygromycin and 0.80 % Bacto agar laid. After incubation at 25 ° C. and 3000 lux for one week, the leaves were inducible to shoot on MS medium with 1.6% glucose, 1.4 mg / 1 zeatin ribose, 20 mg / 1 naphthylacetic acid, 20 mg / 1 giberellic acid, 250 mg / 1 Claforan, 3 mg / 1 hygromycin and 0.80% Bacto Agar. 6. Plant husbandry

Potato plants were kept in the greenhouse under the following conditions:

- Light period 16 h at 25000 lux and 22 ° C

- Dark period 8 h at 15 ° C

- humidity 60%.

Tobacco plants were kept in the greenhouse under the following conditions:

- Light period 14 h at 10000 lux and 25 ° C

- Dark period 10 h at 20 ° C

- Humidity 65%.

7. Determination of acetyl-CoA hydrolase activity in leaves of tobacco and potato plants

To determine the acetyl-CoA hydrolase activity in tobacco and potato leaves, leaf samples were extracted in extraction buffer (50 mM Hepes-KOH pH 7.5; 5 mM MgCl ₂ ; 1 mM EDTA; 1 mM EGTA; 10 mM DTT; 10% ( Vol. / Vol.) Glycerin; 0.1% (Vol. / Vol.) Triton X-100) homogenized. After centrifugation, the cell-free extracts were used for the enzyme activity measurement.

Reaction buffer: 100 mM Na phosphate, pH 7.2

[1- ¹⁴ C] acetyl-CoA 0.5 μCi / 100 μl reaction volume: 105 μl reaction temperature: 30 ° C measuring times: 2, 4, 6, 10 min

Principle:

There are DE81 filters (ion exchanger), where HSCoA and [1- ¹⁴ C] acetyl-CoA bind, however, [1- ¹⁴ C] acetate is eluted in the wash (2 times 5 min), the filter with 2% acetic acid. To determine the acetyl-CoA Hydrolase activity, the radioactivity remaining on the filters was determined by scintillation measurement and comparison with calibration curves.

(see Roughan, P.G. et al., Analytical Biochemistry. 216 (1994), 77-82)

8. Western blot analysis for the mode of expression of acetyl-CoA hydrolase in leaves

To detect acetyl-CoA hydrolase in tobacco and potato leaves, leaf samples were extracted in extraction buffer (50 M Hepes-KOH pH 7.5; 5 mM MgCl ₂ ; 1 mM EDTA; 1 mM EGTA; 10 mM DTT; 10% (vol. / Vol.) Glycerin; 0.1% (Vol. / Vol.) Triton X-100) homogenized. After centrifugation, aliquots (10 μg protein) of the cell-free extracts were used for a Western blot. Western blot analyzes were carried out using a polyclonal antibody directed against the acetyl-CoA hydrolase from Saccharomyces cerevisiae, as described in Landschütze et al. (EMBO J. 14 (1995), 660-666).

9. Determination of metabolic intermediates in tobacco leaves

Sucrose, glucose, fructose and starch were determined spectrophotometrically using coupled enzymatic reactions according to Stitt et al. (Methods in Enzymology, 174, 518-552).

a) Determination of sucrose, glucose and fructose

Three leaf disks each with a diameter of 1.1 cm were extracted with 500 μl 80% ethanol in water over 90 min at 70 ° C. 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 I idazol pH 6.9; 5 mM MgCl ₂ ; 2mM NADP

1 mM ATP

2 U / ml glucose-6-phosphate dehydrogenase

G6P determination: + 1.4 U / ml hexokinase FIP determination: + 1.4 U / ml phosphoglucoisomerase GlP determination: + 2.0 U / ml phosphoglucutase

The measurement is carried out at 30 ° C with 50 μl extract.

(G6P = glucose-6-phosphate; F1P = fructose-1-phosphate;

G1P = glucose-1-phosphate)

b) Determination of strength

Three leaf disks each with a diameter of 1.1 cm were extracted with 500 μl 80% ethanol in water over 90 min at 70 ° C. After separating the solid from the liquid phase by centrifugation, the liquid phase was removed and the solid residue was washed twice with 80% ethanol in water.

The pellet was disrupted with 400 μl of 0.2 N NaOH at 95 ° C. for one hour. Eε was neutralized with 70 μl of 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 range for starch determination (Boehringer, Mannheim) according to the manufacturer's instructions with the aid of amyloglucosidase and the released glucose was determined enzymatically.

c) Determination of fatty acids in leaves and seeds Leaf disks (each 1.1 cm in diameter) or one tobacco seed in 1 ml of 1N HC1 in methanol at 80 ° C under a nitrogen atmosphere after adding 5 μg of meristylic acid as an internal standard for 15 Minutes in a sealed glass jar. After cooling to room temperature, 1 ml of 0.9% aqueous NaCl solution and 1 ml of 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: 141-145 (1986)).

d) Determination of chlorophyll a and b. Antheraxanthin. Zeaxanthin and Violaxanthin

Leaf disks (each 1.1 cm in diameter) were frozen in liquid nitrogen immediately after sampling. The following steps were carried out with the room light darkened.

The samples were homogenized with 250 μl of ice-cold 85% acetone in water, the suspension was then flushed with nitrogen gas for about 30 seconds and then stored on ice for 15 minutes. After centrifugation for 15 minutes at 4 ° C and 10,000 g, the supernatant was filtered through a Millipore-Millex-GV4 sterile filter attachment and then flushed with nitrogen gas at 4 ° C for 30 seconds. The samples were then stored at -70 ° C until measurement. The pigments were separated and quantified by high pressure liquid chromatography (HPLC) as described by Zhayer and Björkman (J. Chromatogr. 543 (1990), 137-145).

A ZORBAX ODS 5 μm non-endcapped 250 * 4.5 mm reversed phase column was used as the separation column. The pigments were detected by measuring the absorption at 450 nm in a range of 0.04 absorption units (AUFS). 10. Determination of citrate synthase activity in leaves of tobacco plants

To determine the citrate synthase activity in tobacco leaves, leaf samples were extracted in extraction buffer (50 mM Hepes-KOH pH 7.5; 5 mM MgCl ₂ ; 1 mM EDTA; 1 mM EGTA; 10% (v / v) glycerin; 0 , 1% (vol. / Vol.) Triton X-100; 4 mU / ml α.2-macroglobulin) homogenized. After centrifugation, the cell-free extracts were used for the enzyme activity measurement.

The activity of the citrate synthase was determined photometrically: Reaction buffer:

0.1 M Tris-Cl pH 8.0

0.1 mM 5, 5 • -dithio-bis (-2-nitrobenzoic acid)

0.3 mM acetyl-CoA reaction volume: 700 μl

Reaction temperature: 30 ° C protein used: ~ 60 μg

Start: 7 μl 50 mM oxaloacetate

The absorption was measured at 412 nm.

The examples illustrate the invention.

example 1

Construction of the binary plasmid Bin-mHy-Int

For the plant transformation, the coding region of the acetyl-CoA hydrolase gene from Saccharomyces cerevisiae was determined using the polymerase chain reaction (PCR) starting from genomic Saccharomyces cerevisiae DNA using the primers AcCoHyl (5 '-GTCAGGATCCATGACAATTTCTAATTTGT IDAAGCAGAGA. 3A)

AcCθHy2 (5 ^» -GTCAGGATCCCTAGTCAACTGGTTCCCAGCTGTCGACCTT-3 ') (Seq ID No. 2) a plified. The sequence of the acetyl-CoA hydrolase from Saccharomyces cerevisiae is entered in the GenEmbl database with the accession number M31036. The cloning of the acetyl-CoA hydrolase gene is described in Lee et al. (Journal of Biological Chemistry 265 (1990), 7413-7418). The amplified fragment corresponds to the region from nucleotides 614 to 2194 of this sequence (accession number M31036). A BamHI interface was inserted at the 5 'end and at the 3' end. The 1590 bp Ba HI cut PCR fragment was cloned into the BamHI site of the vector pUC9-2 via the additional cleavage sites. The intron PIV2 (189 bp) from the plasmid p35S GUS INT (Vancanneyt et al., Mol. Gen. Genet. 220 (1990), 245-250) via PCR using the primer GUS-1 (5 ¹ - gtatacgtaagtttctgcttctac-3 ' ) (Seq ID No. 3) and GUS-2 (5 * - gtacagctgcacatcaacaaattttgg-3 ') (Seq ID No. 4), amplified with SnaBI and PvuII and cut into the unique BbrPI site of the acetyl-CoA hydrolase gene cloned from Saccharomyces cerevisiae. The correct orientation of the inserted intron (5 'end of the intron oriented towards the 3' end of the 5 'located exon) was confirmed by sequence analysis. The plasmid produced in this way received the designation pUC-Hylnt.

The plasmid pUC-Hynt was cut with BamHI and the 1779 bp acetyl-CoA hydrolase fragment (with inserted intron) was cloned into the BamHI site of the plasmid pAM. The plasmid obtained in this way was given the name pAM-Hylnt.

The plasmid pAM was prepared as described below. The mitochondrial target sequence (111 bp) of the protein of the "matrix processing peptidase" (MPP) from potato was determined using the primers Mito-TPl (5'-GATCGGTACCATGTACAGATGCGCATCGTCT-3 ') (Seq ID No. 5) and Mito-TP2 (5 '-GTACGGATCCCTTGGTTGCAACAGCAGCTGA-3') (Seq ID No. 6) amplified by PCR. The plasmid pMPP (Braun et al., EMBO J. 11 (1992), 3219-3227) served as the template for the PCR. The amplified fragment corresponds to the region of the Nucleotides 299 to 397 of the MPP cDNA (Braun et al., See above; EMBL accession number: X66284). An Asp718 interface was inserted at the 5 • end and a BamHI interface at the 3 'end. The PCR fragment was cut with Asp718 and BamHI and the resulting 109 bp fragment was then cloned into the vector pA7 cut with Asp718 and BamHI (from Schaewen, A. (1989) dissertation, Freie Universität Berlin).

The plasmid pAM-Hylnt was cut with Asp718 and Xbal and the 1887 bp fragment consisting of the coding region for the targeting peptide of the potato "Matrix processing peptidase" and the coding region for the acetyl-CoA hydrolase from Saccharomyces cerevisiae (with inserted intron ) isolated and the 5 'overhangs of this fragment filled to blunt ends using T4 DNA polymerase. The fragment thus produced was cloned into the Smal interface of the binary plasmid pBinAR-Hyg. The coding regions of the targeting peptide of the potato "matrix processing peptidase" were oriented towards the 35S RNA promoter of the cauliflower mosaic virus. This resulted in the plasmid Bin-mHy-Int (see FIG. 1), which was used for the transformation of tobacco (Nicotiana tabacu SNN) and potato (Solanum tuberosum L. cv. Desiree) as described above.

Example 2

Construction of the binary plasmid Bin-Hy-Int

The BamHI fragment of the plasmid pUC-Hy-Int (see Example 1) was cloned into the BamHI site of the plasmid pA7 (from Schaewen, A. (1989) dissertation, Free University Berlin). The 5 'end of the coding region of the acetyl-CoA hydrolase was oriented towards the 35S RNA promoter. The plasmid thus produced was given the name pA7-Hy-Int. The plasmid pA7-Hy-Int was cut with Kpnl and Xbal, the 1778 bp fragment consisting of the coding region for the acetyl-CoA hydrolase from Saccharomyces cerevisiae (with inserted intron) and then cloned into the binary plasmid pBinAR-Hyg cut with Kpn ₁ and Xbal (deposit number: DSM 9505; deposit date: 20.10.1994). This resulted in the plasmid Bin-Hy-Int (see FIG. 2), which was used for the transformation of tobacco (Nicotiana tabacum SNN) and potato (Solanum tuberosu L. cv. Desiree).

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 the plasmid Bin-mHy-Int (see Example 1), transferred to soil and by Western blot analysis for the presence of the acetyl-CoA hydrolase from Saccharomyces cerevisiae selected in leaves. Several genotypes were identified which clearly express the Saccharomyces cerevisiae acetyl-CoA hydrolase (see FIG. 3). Several of the selected transgenic lines were analyzed for acetyl-CoA hydrolase activity in leaves. In some lines, a specific acetyl-CoA hydrolase activity increased by up to three times compared to the control plants (e.g. MB-Hyl-39, MB-Hyl-78, MB-Hyl-81; see Table I).

Table I

Plant acetyl-CoA hydrolase activity (n ol min ^" mg ^" protein)

Control 1.49 ± 1.06

MB-Hyl-39 5.58 ± 1.59

MB-Hyl-78 2.92 ± 0.74

MB-Hyl-81 3.25 ± 0.79 The enzyme activities shown here are the mean of at least eight measurements based on at least three independent plants of the transgenic line mentioned.

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

Surprisingly, it was found that the leaves of plants of the transgenic lines MB-Hyl-39, MB-Hyl-78, MB-Hyl-81 contain reduced amounts of fatty acids compared to control plants (cf. Table II).

Table II

FettKontrollMB-Hyl-39 MB-Hyl-78 MB-Hyl-81 acid plant [μmol / g [μmol / g [μmol / g type [μmol / g (dry (dry (dry (dry weight) weight)] weight)] weight) ]

16: 0 2.63 ± 0.35_ 1.17 ± 0.18 2.00 ± 0.34 2.21 ± 0.29

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

Sum: 20.83 ± 2.64 8.17 ± 1.15 15.03 ± 1.78 17.57 ± 1.29

The stated values represent mean values and the standard deviations from 2 independent measurements each.

Surprisingly, it was also found that the seeds of the transgenic plants MB-Hyl-39, MB-Hyl-78, MB-Hyl-81 contain equal amounts of fatty acids compared to control plants (see Table III).

Table III

Fat control MB-Hyl-39 MB-Hyl-78 MB-Hyl-81 acid plant [nmol / seed] [nmol / seed] [nmol / seed] type [nmol / seed]

16: 0 12.28 ± 2.86 10.14 ± 3.90 13.55 ± 2.63 10.71 ± 1.20

16: 1 0.25 ± 0.19 0.27 ± 0.17 0.42 ± 0.09 0.27 ± 0.15

16: 2 0.20 ± 0.17 0.07 ± 0.11 0.09 ± 0.12 0.07 ± 0.08

18: 0 0.02 ± 0.05 0.03 ± 0.06 0.00 ± 0.00 0.01 ± 0.03

16: 3 3.01 ± 1.13 2.04 ± 0.86 3.46 ± 1.12 2.60 ± 0.42

18: 1 13.08 ± 3.49 10.25 ± 5.02 15.00 ± 2.99 10.78 ± 1.37

18: 2 101.49 ± 20.42 86.29 ± 36.05 111.58 ± 19.46 90.03 ± 8.97

18: 3 1.71 ± 0.28 1.74 ± 0.60 2.06 ± 0.35 2.05 ± 0.37

Sum: 132.05 ± 28.59 110.84 ± 46.77 146.16 ± 10.71 116.51 ± 12.59

The values given represent mean values and the standard deviations from at least 6 independent measurements. The analysis of the weight of 200 seeds in each case 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

Seed weight [μg per seed]

Control plant 88.0 ± 6.0

MB-Hyl-39 87.8 ± 2.5

The analysis of soluble sugars such as glucose, fructose and sucrose surprisingly showed that the leaves of Plants of the transgenic lines MB-Hyl-39, MB-Hyl-78, MB-Hyl-81 contain increased levels of sucrose, glucose and fructose both at the end of the light phase (see Table V) and at the end of the dark phase (see Table VI).

Table V

Content of soluble sugars at the end of the light phase

(n = 6)

Glucose fructose sucrose μmol / (dry μmol / g (dry μmol / g (dry weight) weight) weight)

Control 17.3 ± 17.5 31.2 ± 20.0 146.6 ± 54.5 plant

MB-Hyl-39 79.6 ± 44.8 105.8 ± 61.7 294.0 ± 112.7

MB-Hyl-78 188.4 ± 194.2 118.1 ± 129.3 239.9 ± 161.1

MB-Hyl-81 88.6 ± 59.0 24.3 ± 39.5 208.8 ± 148.8

The values given represent mean values and the standard deviations from 6 independent measurements each.

Table VI

Content of soluble sugars at the end of the dark phase (n = 3)

Glucose fructose sucrose μmol / g (dry μmol / g (dry μmol / g (dry weight) weight) weight)

Control 10.2 ± 9.2 5.1 ± 4.4 44.9 ± 13.9 plant

MB-Hyl-39 30.0 ± 18.8 28.1 ± 14.8 150.9 ± 112.3

MB-Hyl -78 142.0 ± 150.3 53.4 ± 30.2 190.9 ± 200.8

MB-Hyl-81 8.8 ± 7.6 12.9 ± 1.9 16.5 ± 2.1 The stated values represent mean values and the standard deviations from 3 independent measurements each.

In the analysis of starch, it was also surprisingly found that the leaves of plants of the transgenic lines MB-Hyl-39, MB-Hyl-78, MB-Hyl-81 increased levels of starch both at the end of the light phase and at the end of the Dark phase included (see Table VII).

Table VII

Maintain strength at the end of the light phase and at the end of the

Dark phase

End of light phase - end of dark phase εe (n = 6) (n = 3) mmol / g (dry weight) mmol / g (dry weight)

Control 2.37 ± 0.67 0.97 ± 0.76 plant

MB-Hyl-39 4.81 ± 1.82 2.57 ± 2.08

MB-Hyl-78 1.79 ± 1.49 1.77 ± 2.18

MB-Hyl-81 2.23 ± 1.33 0.17 ± 0.02

The values given represent mean values and the standard deviations from 3 or 6 (see Table VII) independent measurements.

When the transgenic plants MB-Hyl-39, MB-Hyl-78, MB-Hyl-81 were grown in the greenhouse, it was also found that the transgenic plants had a different phenotype compared to control plants. In particular, in the case of the transgenic plants, reduced growth, the formation of several shoots and a mosaic-like change in the leaf color were found (see FIGS. 4 and 5). For a more precise analysis of the leaf color, the levels of chlorophyll a and b, as well as of the carotenoids Zeaxanthin, antheraxanthin and violoxanthin determined (see table VIII and IXa and b).

Table VIII

Chlorophyll b content Chlorophyll content a

(μmol / g (μmol / g

(Dry weight) (dry weight)

Control 5.42 ± 0.46 5.98 ± 0.54 plant

MB-Hyl-39 2.38 ± 0.21 2.73 ± 0.21

MB-Hyl-78 4.23 ± 0.82 4.81 ± 0.74

MB-Hyl-81 5.13 ± 0.36 6.03 ± 0.54

The values given represent mean values and the standard deviations from 3 independent measurements each.

Table IXa

Violaxanthin Antheraxanthin Zeaxanthin (μmoϊ / g (μmol / g (μmol / g (dry weight (dry weight (dry weight) weight) weight) weight)

Control 0.173 ± 0.042 0.178 ± 0.049 0.299 ± 0.114 plant

MB-Hyl-39 0.215 ± 0.045 n.d. n.d. n.d. n.d.

MB-Hyl-78 0.314 ± 0.052 0.051 ± 0.036 n.d. n.d.

MB-Hyl-81 0.591 ± 0.205 0.034 ± 0.004 n.d. n.d.

nd not detected (below the detection limit) Table IXb

Lutein neoxanthin

(μmol / g (μmol / g

(Dry weight) (dry weight)

Control plant 1.94 ± 0.32 0.38 ± 0.06

MB-Hyl-39 0.74 ± 0.11 0.15 ± 0.03

MB-Hyl-78 1.46 ± 0.51 0.22 ± 0.02

MB-Hyl-81 1.79 ± 0.40 0.39 ± 0.11

The stated values represent mean values and the standard deviations from 3 independent measurements each.

Example 4

Construction of the binary plasmid pTCSAS

The in vivo excision from a λ ZAP II cDNA library from Nicotiana tabacum L. Plasmid pTCS obtained contains a 1747 bp cDNA fragment with the coding region of the citrate synthase gene from tobacco (accession number X84226) in the EcoRI interface of the pBluescript SK vector. The BamHI fragment of the plasmid pTCS was cloned into the BamHI / SalI sites of the binary plasmid BinAR. The 3 'end of the coding region of the citrate synthase was oriented towards the 35S RNA promoter. The plasmid thus produced was given the name 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 which had been transformed with the plasmid pTCSAS were transferred into 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 citrate synthase activity in leaves. In some lines, a specific citrate synthase activity reduced by up to six times compared to the control plants was measured (e.g. TCSAS-14, TCSAS-17; TCSAS-26; TCSAS-43; TCSAS-48; see Table X).

Table X

CS = C trate synthase

The enzyme activities shown here are the average of at least 18 measurements 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.

Surprisingly, it was found that the seeds of the transgenic plants TCSAS-17 and TCSAS-26 contain reduced amounts of fatty acids compared to control plants (cf. Table XI).

Table XI

wt t 17 t 26 [nmol / seed] [nmol / seed] [nmol / seed]

16.: 0 15.42 ± 2.96 7.03 ± 1.74 9.58 ± 1.37

18.: l 17.78 ± 4.12 6.88 ± 2.02 9.24 ± 2.29

18.: 2 116.27 ± 17.66 63.25 ± 12.09 80.13 ± 14.74

18.: 3 1.74 ± 0.38 1.23 ± 0.27 1.44 ± 0.27

Sum: 151.22 ± 25.11 78.39 ± 16.13 100.39 ± 18.68

The values given represent mean values and the standard deviations from 10 independent measurements in each case.

The 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 compared to the seeds of control plants (cf. Table XII).

Table XII

Example 6

Construction of the plant transformation vector pBin-USP-

MTPHylnt

For the plant transformation, the coding region of the acetyl-CoA-Hvdrolase gene from Saccharomyces cerevisiae was isolated by means of the polymerase chain reaction (PCR) as described in Example 1.

The plasmid pAM-Hylnt was cut with Asp718 and Hindlll and the 1931 kb fragment consisting of the coding region of the targeting peptide of the potato "Matrix processing peptidase" and the coding region for the acetyl-CoA hydrolase from Saccharomyces cerevisiae (with inserted intron) was isolated. The fragment isolated in this way was cloned into the Asp718 / HindIII cleavage sites of the binary plasmid pUSP-Bin19. This vector contains the USP promoter (Fiedler et al., Plant Mol. Biol. 22 (1993), 669-679), in particular the 684 bp Pstl fragment from pP30T, a camycin resistance gene, a multiple cloning gene Site and the polyadenylation site of the octopine synthase gene. The resulting plasmid pBin-USP-MTPHylnt was used for the transformation of oilseed rape as described above.

Example 7

Construction of the plant transformation vector pBin-

USP / Hyg-TP-ACS

For the plant transformation, the coding region of the acetyl-CoA proteinase gene (De Virgilio et al., Yeaεt 8 (1992), 1043-1051) from Saccharomyces cerevisiae was determined using the polymerase chain reaction (PCR) starting from genomic S. cerevisiae DNA using the Primers ACS1 (5 ^» GAT CAA GCT TAT GTC GCC CTC TGC CGT ACA ATC -3 ^• ; Seq ID No. 7) and ACS2 (5'- GAT CAA GCT TTC ATC ATT ACA ACT TGA CCG ATC C-3 •, Seq ID No. 8) amplified. The sequence of the acetyl-CoA synthetase is entered in the GenEMBL database with the access number X66425. The cloning of the acetyl-CoA synthetase gene is described in De Virgilio (loc. Cit.). The amplified fragment corresponds to the region from nucleotides 162 to 2303 of this sequence. Here, a HindIII interface was inserted at the 5 'end and at the 3' end. The 2151 bp HindIII fragment was cloned into the HindIII site of the vector pSK-TP via the additional cleavage sites. This plasmid contains a DNA sequence which encodes the plastid transit peptide of ferredoxin: NADP ⁺ oxireductase from spinach. The plasmid pSK-TP-ACS was cut with Asp718, the interfaces filled in with the help of T4-DNA polymerase to a smooth end and then cut Xbal. The 2380 kb fragment thus isolated, consisting of the coding region of the targeting peptide of spinach ferrodoxin: NADP ⁺ oxireductase and the coding region for the acetyl-CoA synthetase from S. cerevisiae was cloned into the Smal / Xbal interfaces of the binary plasmid pBin-USP-Hyg. This vector contains the USP promoter (684 Pεtl fragment from pP30T (Fiedler et al., Loc. Cit.). The resulting plasmid pBin-USP / Hyg-TP-ACS was used for the transformation of oilseed rape plants that use an acetyl-CoA hydrolase Express yeast (see Example 4) as used above.

Example 8

Construction of the plant transformation vector pBin-

USP / Hyg-TP-ACLY

For the plant transformation, the coding region of the ATP: citrate lyase gene from Rattus norvegicus was determined using the polymerase chain reaction (PCR) starting from cDNA from Rattus norvegicus using the primer ACLY1 (5'-ACT GAA GCC TAT GTC AGC CAA GGC AAT TTC AGA GCA -3 ', Seq ID No. 9) and ACLY2 (5'- ACT GAA GCC TTT ACA TGC TCA TGT GTT CCG GGA GAA C -3', Seq ID No. 10). The sequence of the ATP: citrate lyase is in the GenEMBL database with the accession number J05210 registered. The cloning of the ATP: citrate lyase gene from Rattus norvegicus is described in Elshourbagy et al. (J. Biol. Chem. 265 (1990), 1430-1435). The amplified fragment corresponds to the region from nucleotides 73 to 3375 of this sequence. Here, a HindIII interface was inserted at the 5 'end and at the 3' end. The 3312 bp HindIII fragment was cloned via the additional interfaces into the HindIII interface of the vector pSK-TP. The plasmid pSK-TP-ACLY was cut with Asp718, filled in with blunt ends using T4 DNA polymerase and then cut with Xbal. The 3494 kb fragment isolated in this way, consisting of the coding region of the targeting peptide of spinach ferredoxin: NADP ⁺ oxireductase and the coding region for the ATP: citrate lyase from Rattus norvegicus, was inserted into the Smal / Xbal interfaces of the binary plasmid pBin-USP -Hyg cloned. The resulting plasmid pBin-USP / Hyg-TP-ACLY was used for the transformation of oilseed rape as described above.

Example 9

Construction of the plant transformation vector pBin-USP-

MTPCS

The coding region of the citrate protein gene from E. coli using the polymerase chain reaction (PCR) starting from genomic JE., coli DNA using the primers CS1 (5'- A CTG GGA TCC ATG GCT GAT ACA AAA 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) a plified. The cloning and sequence of the citrate synthase gene is described in Ner et al., Biochemistry 22 (1983), 5243-5249. The amplified fragment corresponds to the coding region from nucleotides 1 to 1284 of this sequence. A BamHI interface was inserted at the 5 'end and at the 3' end. The 1294-long BamHI fragment was inserted into the BamHI section via the additional cleavage sites. site of the vector pAM cloned. The plasmid pAM-CS was cut with Asp718 and HindIII and filled in to blunt ends with the T4-DNA polymerase, and the 1393 kb fragment consisting of the coding region of the targeting peptide of the potato "matrix processing peptidase" and the coding region for the citrate synthase from E. coli isolated. The fragment isolated in this way was cloned into the Smal sites of the binary plasmid pUSP-Bin19 (see Example 6). The resulting plasmid pBin-USP-MTPCS was used on the one hand for the transformation of tobacco plants and on the other hand of rapeseed plants as described above.

Example 10

Transgenic plants with a mitochondrial acetyl-CoA hydrolase and a plastid acetyl-CoA synthetase

Plants that co-express an acetyl-CoA hydrolase from yeast with mitochondrial targeting and an acetyl-CoA synthetase from yeast with plastid targeting were regenerated and grown in the greenhouse. The seeds of these plants were examined for their total fatty acid content as described above. An increase of approx. 5% with respect to the total fatty acid content (per seed) was found in comparison to non-transformed plants.

Table XIII

Line fatty acid content Δ fatty acid content [nmol / seed]

Control 133 11 tl 144 12 t2 147 28 t3 145 17 t4 143 18 t5 146 12 SEQUENCE LIST (1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Max Planck Society for the Promotion of

Sciences e.V.

(B) ROAD: none

(C) LOCATION: Berlin

(E) COUNTRY: Germany

(ii) NAME OF THE INVENTION: Transgenic plant cells and plants with altered acetyl-CoA formation

(iii) NUMBER OF SEQUENCES: 12

(iv) COMPUTER READABLE VERSION:

(A) DISK: Floppy disk

(B) COMPUTER: IBM PC compatible

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

(D) SOFTWARE: Patentin Release # 1.0, Version # 1.30 (EPA)

(2) INFORMATION ON SEQ ID NO: 1:

(i) SEQUENCE LABEL:

(A) LENGTH: 40 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Other nucleic acid

(A) DESCRIPTION: / desc = "oligonucleotides"

(iii) HYPOTHETICAL: YES

(iv) ANTISENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GTCAGGATCC ATGACAATTT CTAATTTGTT AAAGCAGAGA 40

(2) INFORMATION ON SEQ ID NO: 2:

(i) SEQUENCE LABEL:

(A) LENGTH: 40 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Other nucleic acid

(A) DESCRIPTION: / desc = "Oligonucleotides ¹ (iii) HYPOTHETICAL: YES (iv) ANTISENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: GTCAGGATCC CTAGTCAACT GGTTCCCAGC TGTCGACCTT 40

(2) INFORMATION ON SEQ ID NO: 3:

(i) SEQUENCE LABEL:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Other nucleic acid

(A) DESCRIPTION: / desc = "oligonucleotide"

(iii) HYPOTHETICAL: YES

(iv) ANTISENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: GTATACGTAA GTTTCTGCTT CTAC 24

(2) INFORMATION ON SEQ ID NO: 4:

(i) SEQUENCE LABEL:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Other nucleic acid

(A) DESCRIPTION: / desc = "oligonucleotide"

(iii) HYPOTHETICAL: YES

(iv) ANTISENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: GTACAGCTGC ACATCAACAA ATTTTGG 27 (2) INFORMATION ON SEQ ID NO: 5:

(i) SEQUENCE LABEL:

(A) LENGTH: 31 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Other nucleic acid

(A) DESCRIPTION: / desc = "oligonucleotide"

(iii) HYPOTHETICAL: YES

(iv) ANTISENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: GATCGGTACC ATGTACAGAT GCGCATCGTC T 31

(2) INFORMATION ON SEQ ID NO: 6:

(i) SEQUENCE LABEL:

(A) LENGTH: 31 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Other nucleic acid

(A) DESCRIPTION: / desc = "oligonucleotide"

(iii) HYPOTHETICAL: YES

(iv) ANTISENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: GTACGGATCC CTTGGTTGCA ACAGCAGCTG A 31

(2) INFORMATION ON SEQ ID NO: 7:

(i) SEQUENCE LABEL:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Other nucleic acid

(A) DESCRIPTION: / desc = "oligonucleotide" (iii) HYPOTHETICAL: YES (iv) ANTISENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: GATCAAGCTT ATGTCGCCCT CTGCCGTACA ATC 33

(2) INFORMATION ON SEQ ID NO: 8:

(i) SEQUENCE LABEL:

(A) LENGTH: 34 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Other nucleic acid

(A) DESCRIPTION: / desc = "oligonucleotide"

(iii) HYPOTHETICAL: YES

(iv) ANTISENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: GATCAAGCTT TCATCATTAC AACTTGACCG ATCC 34

(2) INFORMATION ON SEQ ID NO: 9:

(i) SEQUENCE LABEL:

(A) LENGTH: 36 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Other nucleic acid

(A) DESCRIPTION: / desc = "oligonucleotide"

(iii) HYPOTHETICAL: YES

(iv) ANTISENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: ACTGAAGCCT ATGTCAGCCA AGGCAATTTC AGAGCA 36 (2) INFORMATION ON SEQ ID NO: 10:

(i) SEQUENCE LABEL:

(A) LENGTH: 37 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Other nucleic acid

(A) DESCRIPTION: / desc = "oligonucleotide"

(iii) HYPOTHETICAL: YES

(iv) ANTISENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: ACTGAAGCCT TTACATGCTC ATGTGTTCCG GGAGAAC 37

(2) INFORMATION ON SEQ ID NO: 11:

(i) SEQUENCE LABEL:

(A) LENGTH: 38 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Other nucleic acid

(A) DESCRIPTION: / desc = "oligonucleotide '

(iii) HYPOTHETICAL: YES

(iv) ANTISENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: ACTGGGATCC ATGGCTGATA CAAAAGCAAA ACTCACCC 38

(2) INFORMATION ON SEQ ID NO: 12:

(i) SEQUENCE LABEL:

(A) LENGTH: 35 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Other nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: ACTGGGATTC TTAACGCTTG ATATCGCTTT TAAAG 35

Claims

Patent claims

1. Transgenic plant cell with an altered acetyl-CoA metabolis us which, due to the expression of a foreign DNA sequence encoding a protein with acetyl-CoA hydrolase activity, has an increased acetyl-CoA hydrolase activity compared to wild-type cells.

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

3. Transgenic plant cell according to claim 2, which further has an increased 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 increased.

5. Transgenic plant cell according to claim 2 or 4, which further has an increased acetyl-CoA synthetase activity in the plastids.

6. Transgenic plant cell according to one of claims 1 to 5, which further has a reduced citrate synthase activity in the mitochondria.

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

8. Transgenic plant cell according to one of claims 1 to 7, which further has a reduced activity of the ATP citrate lyase in the cytosol.

9. Transgenic plant cell according to one of claims 1 to 7, which further has an increased activity of the ATP citrate lyase in the cytosol.

10. Transgenic plant cell according to 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 is derived from fungal cells.

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

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

14. Transgenic plant cell according to 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 is derived from a fungus, a bacterial or an animal organism.

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

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

18. Plant according to claim 17, which has at least one of the following features:

(c) an increased starch content in leaf tissue compared to wild-type plants;

(d) reduced growth;

(e) formation of two or more rungs;

(f) change in leaf color.

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

20. Plant propagation material according to one of claims 17 to 19, which contains transgenic plant cells according to 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. Use according to claim 22, wherein the acetyl-CoA hydrolase activity in the cytosol is increased.