WO1997007221A1

WO1997007221A1 - Transgenic plant cells and plants having an increased glycolysis rate

Info

Publication number: WO1997007221A1
Application number: PCT/EP1996/003514
Authority: WO
Inventors: Jörg Riesmeier; Richard Trethewey; Lothar Willmitzer
Original assignee: Planttec Biotechnologie Gmbh
Priority date: 1995-08-11
Filing date: 1996-08-08
Publication date: 1997-02-27
Also published as: CN1196090A; BR9610227A; CA2229061A1; AU719452B2; EP0846180A1; JP2001506123A; DE19529696A1; AU6820496A

Abstract

The invention concerns transgenic plant cells and plants having an increased glycolysis rate. The glycolysis rate is increased by the introduction and expression in plant cells of a DNA sequence which codes for an invertase, preferably a deregulated or unregulated invertase, and a DNA sequence which codes for a hexokinase, preferably a deregulated or unregulated hexokinase. The invention further concerns processes and recombinant DNA molecules for producing plant cells and plants having an increased glycolysis rate.

Description

Transgenic plant cells and plants with increased

Glycolysis rate

The present invention relates to uransgenic plant cells and plants with a higher rate of glycolysis compared to non-transformed plants. The increase in the rate of glycolysis is achieved by introducing and expressing a DNA sequence encoding a cytosolic invertase, preferably a deregulated or unregulated invertase, and a DNA sequence encoding a cytosolic hexokinase, preferably a deregulated or unregulated hexokinase encoded, in plant cells. The invention also relates to methods and recombinant DNA molecules for the production of transgenic plant cells and plants with an increased glycolysis rate, and to the use of DNA sequences which contain proteins with the enzymatic activity of an invertase or proteins with the enzymatic activity encode a hexokinase for the production of plants which have an increased rate of glycolysis.

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 useful plants. One way to achieve this is to specifically change the metabolism of plants by genetic engineering. The goals are, for example, the primary processes of photosynthesis that lead to CO ₂ fixation, the transport processes that are involved in the distribution of the photoassimilates within the plant, and metabolic pathways that lead to the synthesis of storage substances, eg starch, Proteins or oils lead. For example, the expression of a prokaryotic asparagus has been described. gin synthetase in plant cells, which, among other things, leads to an increase in biomass production in the transgenic plants (5P-3 0 511 979). The expression of a prokaryotic polyphosphate kinase in the cytosol of transgenic plants has also been proposed, as a result of which an increase in the yield of the tuber weight by up to 30% in potato plants. Furthermore, EP-A2 0 442 592 describes the expression of an apoplastic invertase in potato plants, which likewise leads to an increase in the yield of such modified transgenic plants. Further attempts focused on modifying the activities of enzymes which are involved in the synthesis of sucrose as the most important transport metabolite in most plants (cf. for example Sonnewald et al., Plant Cell and Environment 17 (1995), 649- 658). WO 91/19896 further describes that the increase in starch biosynthesis by overexpression of a deregulated enzyme of ADP-glucose pyrophosphorylase leads to an increased allocation of sucrose in potato tubers, which are stored there in the form of starch. While many of these applications deal with the steps which either lead to the formation of photoassimilates in leaves (cf. also EP 466 995) or with the formation of polymers such as starch or fructans in storage organs of transgenic plants (for example 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 achieve an increase in the rate of glycolysis. An increase in the rate of glycolysis is important, for example, for all those processes in the plant for which larger amounts of ATP are required. This applies, for example, to many transport processes across membranes that are driven by a membrane potential or a proton gradient that is generated by the activity of an H ⁺ -ATPase. Furthermore, an increase in the rate of glycolysis is important for growth in the meristem, the switch from vegetative to generative Growth, as well as for the synthesis of various storage materials, in particular oils.

The invention is therefore based on the object of making available plant cells and plants with an increased glycolysis rate as well as processes and DNA molecules for their production.

This object is achieved by providing the embodiments described in the patent claims.

The present invention thus relates to transgenic plant cells with a glycolysis rate which is increased in comparison to non-transformed plant cells, in which it is due to the introduction and expression of DNA sequences which code for a cytosolic invertase, and of DNA sequences, encoding a cytosolic hexokinase leads to an increase in invertase and hexokinase activity. The transgenic cells can each contain one or more DNA sequences which encode an invertase or a hexokinase.

It was surprisingly found that the introduction and expression of DNA sequences which encode a protein with the enzymatic activity of a hexokinase, with the simultaneous introduction and expression of DNA sequences, of a protein with the enzymatic activity of an invertase encode, in the cytosol of plant cells, a drastic increase in the rate of glycolysis in plant cells modified in this way can be achieved in comparison with plant cells which have not been modified. The expression of an invertase of fungal origin in the cytosol has already been described (cf. EP-A2 442 592). However, only the expression of the fungal invertase in the cytosol of plant cells alone has been described. The use of cytosolic invertase to increase the rate of glycolysis was not described, in particular especially in combination with the expression of a hexokinase.

An increased glycolysis rate means that the transgenic plant cells which have been transformed with DNA sequences which lead to the additional synthesis of an invertase and a hexokinase in the cytosol of the cells have an increased glycolysis rate, preferably a glycolysis rate, compared to non-transformed plant cells which is increased by at least 30%, in particular one which is increased by at least 50% to 100%, and in particular one which is increased by more than 100% to 200%, preferably by more than 300%. The determination of the glycolysis rate by determining the corresponding metabolic intermediates is described in detail in the examples. The increase in the glycolate can be determined, for example, by increasing the concentration of glucose-6-phosphate, fructose-6-phosphate, 3-phosphoglycerate, pyruvate or ATP. In the context of the present invention, the increase in the rate of glycolysis is preferably determined by determining the increase in pyruvate.

The increase in the rate of glycolysis is compared. _. determined with non-transformed cells. This means that material from plants which were used as the starting material for the introduction of the above-mentioned DNA sequences is used to determine the glycolysis rate and the glycolysis rate determined in this is compared with that of plants corresponding type or line after the transformation with the DNA sequences described above.

The provision of larger amounts of ATP through an increased glycolysis rate enables, for example, the increase in various energy-dependent transport and growth processes. The provision of higher pyruvate concentrations as a result of an increased glycolysis rate leads to increased amounts of AcetylCoA, which is used, for example, for the increased synthesis of oils, fats or isoprenoid derivatives can be. If DNA sequences which enable the synthesis of polyhydroxyalkanoic acids are simultaneously expressed in the cells, the acetylCoA can also be used to form such alkanoic acids.

In a preferred embodiment of the invention, the DNA sequences which encode an invertase or a hexokinase are DNA sequences which encode an invertase or hexokinase, in comparison to invertases or hexoki which normally occur in plant cells ¬ noses are deregulated or unregulated. Deregulated means that these enzymes are not regulated in the same way as the invertase and hexokinase 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 invertases or hexokinases expressed in plant cells. Unregulated _means in the context of this invention that the enzymes in plant len Zel¬ no regulation subject.

The DNA sequences which encode a protein with the enzymatic activity of an invertase can be both DNA sequences which encode prokaryotic, in particular bacterial, invertases and those which eukaryotic, ie invertases from plants, algae, Coding mushrooms or animal organisms. In the context of this invention, the term fungi also includes yeasts, in particular those of the genus Saccharomyces, such as, for example, Saccharomyces cerivisiae. The enzymes encoded by the sequences can be either known enzymes occurring in nature which have different regulation by various substances, in particular by the plant invertase inhibitors, or enzymes which by mutagenesis of DNA sequences which encode known enzymes from bacteria, algae, fungi, animals or planters.

In a preferred embodiment of the present invention, the DNA sequences encode proteins with the enzymatic activity of an invertase from fungi. Such enzymes have the advantage that they are not regulated by plant invertase inhibitors in comparison to plant invertases. DNA sequences encoding an invertase from Saccharomyces are preferably used. Such sequences are known and described (see. Taussig et al., Nucleic Acids Res. 11 (1983), 1943-1954; EP-A2 0 442 592). In order to ensure the localization of the invertase in the cytosol of the plant cells, existing DNA sequences that code for signal peptides may have to be deleted and the coding region may have to be provided with a new start codon (cf. EP-A2 0 442 592). In addition to the DNA sequence mentioned from Saccharomyces cerevisiae, other DNA sequences are also known which code proteins with the enzymatic activity of an invertase (cf. EMBL Accession number X67744 S. xylosus, M26511 V. alginolyticus, L08094 Zymomonas mobilis, L33403 Zymomonas mobilis, U16123 Zea mays, U17695 Zea mays, X17604 S. occidentalis, Z22645 S. tuberosum, Z21486 S. tuberosum, M81081 tomato, Z35162 V. faba, Z35163 V. faba, D10265 V. radiata, Z12025 L. esculentum, Z12028 pimpinellifolium, Z12026 L. pimpinellifolium, X73601 A. sativa, S70040 acid invertase, V01311 yeast gene, U11033 Arabidopsis thaliana, X81795 B. vulgaris BIN35, X81796 B. vulgaris, X81797 B. vulgaris, X81793 C. rubrum, X81792 C. rubrum, , X77264 L. esculentum, Z12027 L. esculentum, D10465 Z. mobilis, D17524 Zymomonas mobilis) and which due to their properties can also be used for the production of the plant cells according to the invention, care being taken that the P rotein in the cytosol of the plant cell is formed. Techniques for Modifying Such DNA Se- sequences to localize the synthesized. Ensuring enzymes in the cytosol of the plant cells is known to the person skilled in the art. In the event that the invertases 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.

The DNA sequences which encode a procyne with the enzymatic activity of a hexokinase can be both those which encode prokaryotic, in particular bacterial, invertases and those which encode eukaryotic invertases, i.e. encode those from plants, algae, fungi or animal organisms.

Hexokinases (EC 2.7.1.1) are enzymes that catalyze the following reaction:

Hexose + ATP <—> Hexose-Phosphate + ADP The hexokinases encoded by the DNA sequences can be known, naturally occurring enzymes, which have different regulation by different substances, as well as enzymes, which by mutage ¬ nese from DNA sequences that encode known enzymes from bacteria, algae, fungi, animals or plants. DNA sequences which encode enzymes with hexokinase activity have been described from a whole series of organisms, for example from Saccharomyces cerevisiae, humans, rats and various microorganisms (for the DNA sequences see: EMBL gene bank access numbers M92054, LO4480, M65140 , X61680, M14410, X66957, M75126, J05277, J03228, M68971, M86235, X63658).

In a preferred embodiment, these are DNA sequences which code for glucokinases, in particular glucokinases, which are subject to reduced allosteric regulation, for example by glucose-6-phosphate. Glucokinases (EC 2.7.1.2) are hexokinases with a high affinity for glucose, which catalyze the following reaction: Glucose -r ATP <—> glucose-6-phosphate + ADP

In a preferred embodiment, the DNA sequences encode a giucokinase from Zymomonas mobilis (Barneil et al., J. Bacteriol. 172 (1990), 7227-7240; EMBL gene bank access number M60615). Further glucokinases from humans and rats have been described (for the DNA sequences see: EMBL gene bank accession numbers M69051, M90299, J04218 and M25807).

Furthermore, DNA sequences which encode an invertase or a hexokinase can be isolated from any organism with the aid of the already known DNA sequences mentioned above. 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. Methods for determining the invertase or hexokinase activities are familiar to the person skilled in the art.

By introducing mutations and modifications according to techniques known to the person 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.

For expression in plant cells, the DNA sequences which encode a cytosolic invertase or hexokinase can in principle be under the control of any promoter which is 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, but is preferably found in such tissues instead, in which an increased glycolysis rate is advantageous either for the growth of the plant, for the uptake and transport of ions and metabolites or for the formation of ingredients within the plant. Promoters which ensure specific expression in a specific tissue, at a specific development time of the plant or in a specific organ of the plant therefore appear to be particularly suitable. Promoters which are specifically active in the endosperm or in the cotyledons of seeds forming therefore appear to be particularly suitable for increasing the fatty acid biosynthesis as a result of an increased acetylCoA content in seeds of oil-forming plants such as oilseed rape, soybean, sunflower and oil palms. Such promoters are, for example, the Phaseolin promoter from Phaseolus vulgaris, the USP promoter from Vicia faba or the HMG promoter from wheat.

It is also advantageous to use promoters that ensure seed-specific expression. In the case of starch-storing plants, e.g. This increases the rate of glycolysis in the seeds of maize, wheat, barley or other cereals, and there is an increased formation of pyruvate and acetylCoA and an increased fatty acid biosynthesis. This means that a change in the flow of photoassimilates from starch towards pyruvate-dependent biosynthetic pathways, e.g. fatty acid biosynthesis.

It is also preferred to use promoters which are active in storage organs such as tubers or roots, e.g. in the root of the sugar beet or in the tuber of the potato. In this case, the expression of the DNA sequences encoding an invertase or hexokinase leads to a redirection of biosynthetic pathways in the sense of the formation of less sugar or starch and an increased formation of pyruvate and acetyl-CoA due to the increased rate of glycolysis.

Furthermore, the expression of the DNA sequences can take place under the control of promoters, which are specific at the time the flowering induction are activated or are active in tissues that are necessary for the flowering induction. Promoters can also be used which are controlled only by external influences. Be activated at the time, for example by light, temperature, chemical substances (see, for example, WO 93/07279). For increasing the rate of uptake of ions from the soil as a result of an increased ATP content, promoters are of interest, for example, which have root hair or root epidermis-specific expression. To increase the export rate of photoassimilates from the leaf, promoters are of interest, for example, which have a transmission cell-specific expression. Such promoters are known (for example the promoter of the rolC gene from Agrobacterium rhizogenes).

Furthermore, the DNA sequences which encode an invertase or hexokinase can, apart from a promoter, advantageously be linked 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 area and which ensure a more efficient translation of the synthesized RNA into the corresponding protein (so-called translation enhancer). 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 DNA sequences which code for an invertase or a hexokinase are linked to 3 ¹ - non-translated 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 encode an invertase or hexokinase are preferably stably integrated into the genome in the plant cells according to the invention. The transgenic plant cells which, owing to the additional expression of a cytosolic invertase and a cytosolic hexokinase, have an increased rate of glycolysis, can in principle be cells of any plant species. Of interest are both cells of monocotyledonous and dicotyledon plant species, in particular cells of starch-storing, oil-storing or agricultural useful plants, such as, for example, rye, oats, barley, wheat, potato, corn, rice, rapeseed, peas, sugar beet, Soybean, tobacco, cotton, sunflower, oil palm, wine, tomato etc. or cells of ornamental plants.

In addition to an increased glycolysis rate, the plant cells according to the invention can be distinguished from corresponding non-transformed plant cells in that they contain foreign DNA sequences which are stably integrated into the genome and which encode a cytosolic invertase or a cytosolic hexokinase. In this context, the term “foreign DNA sequence” means the following: on the one hand, it can be DNA sequences that are heterologous with respect to the transformed plant cell, i.e. do not naturally occur in such a plant cell. If the DNA sequences are those which naturally occur in the transformed plant cells, "foreign" means that they are integrated in the genome of the transformed plant cells at a location where they do not occur naturally. ie they are in a new genomic environment. This can be verified, for example, by a Southern blot analysis. Furthermore, the DNA molecules introduced into the plant cells are generally recombinant DNA molecules, i.e. molecules that are composed of different segments that do not occur naturally in this combination.

The present invention furthermore relates to transgenic plants which contain transgenic plant cells according to the invention. hold. Such plants can be generated, for example, by regeneration from a plant cell according to the invention.

By providing plant cells with an increased glycolysis rate, it is now possible to produce transgenic plants with changed advantageous properties. Thus, for example, by increasing the rate of glycolysis specifically in transgenic cells of transgenic plants, the loading of the sieve element-condenser cell complex with sucrose can be increased by the sucrose-proton co-transporter, which leads to an increase in the transport rate of photoassimilates. In the same way, the absorption of inorganic ions such as phosphate, sulfate, nitrate, etc. from the soil can be increased via the root. An increase in the rate of glycolysis specifically in root cells, in particular in root hairs and epidermal cells, can lead to an increased excretion of protons into the soil due to the increased H ⁺ -ATPase activity. Such acidification of the soil leads to mobilization and thus easier absorption of various minerals, such as phosphate from the soil.

Of particular importance is the possibility, by increasing the rate of glycolysis in oil-storing tissues of a plant, such as, for example, 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 the formation of pyruvate and Steer AcetylCoA. The provision of increased amounts of these precursors for the biosynthesis of triglycerides leads to an increased synthesis of oils and thus to an increased yield. The provision of increased amounts of AcetylCoA is also important for many other processes that occur naturally in plants, such as isoprenoid biosynthesis, but also for the formation of polymers such as polyhydroxyalkanoic acids (see, for example, Poivier et al., Bio / Technology 13 ( 1995), 142-150). An increase in the rate of glycolysis in starch-storing tissues, for example in seeds of the most varied types of cereals or in the tubers of the potato, leads to a change in the flow of photoassimilates from starch biosynthesis towards pyruvate-dependent biosynthetic pathways, for example fatty acid biosynthesis. The result is a reduction in the amount of starch in the corresponding tissue with a possible simultaneous increase in the fatty acid biosynthesis.

It is also important that the high acetylCoA concentrations generated by increasing the glycolysis rate can also lead to increased isoprenoid synthesis or, in combination with the expression of appropriate genes for the synthesis of polyhydroxyalkanoic acids, to polyhydroxyaicanoic acid synthesis in the transgenic plant cells .

Furthermore, the present invention relates to a method for producing transgenic plant cells which have an increased glycolysis rate in comparison to non-transformed plant cells, in which DNA sequences which encode a cytosolic invertase are introduced into plant cells, and also DNA sequences , which encode a cytosolic hexokinase, and these sequences are expressed in the transformed plant cells.

Such a method preferably consists of the following steps:

(a) Production of a recombinant double-stranded DNA molecule which comprises the following DNA sequences:

(i) a promoter which ensures transcription in plant cells;

(ii) a DNA sequence which encodes a cytosolic protein with the enzymatic activity of an invertase and is linked in the sense direction to the promoter;

(b) the production of a recombinant double-stranded DNA molecule which comprises the following DNA sequences: 1 Δ.

(i) a promoter which ensures transcription in plant cells; (ii) a DNA sequence which codes a cytosolic protein with the enzymatic activity of a hexokinase and is linked in the sense direction to the promoter; and (c) transfer of the DNA molecules produced according to step (a) and (b) into plant cells.

For the selection of the plant species, the promoters, further flanking DNA sequences, and for the selection and modifications of the DNA sequences which encode an invertase or a hexokinase, the same applies as in connection with the cells according to the invention was carried out.

The DNA sequences which encode an invertase or a hexokinase can either be located on separate DNA molecules or together on a recombinant DNA molecule. If the sequences are on two different DNA molecules, the transfer of the DNA molecules can either take place simultaneously or in such a way that plant cells are first transformed with a DNA molecule and then selected plant cells or plants are subsequently transformed with the second DNA molecule become. Furthermore, plants which express both an additional cytosolic invertase and an additional cytosolic hexokinase can be produced by first generating two independent transgenic plant lines which code for an invertase or a hexokinase and then crossing them.

The transfer of the DNA molecules which contain DNA sequences which encode invertase or hexokinase is preferably carried out using plasmids, in particular those plasmids which ensure stable integration of the DNA molecule into the genome of transformed plant cells, with so binary binary plasmids or Ti plasmids of the Agrobacterium tumefaciens system. In addition to the Agrobacterium 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, 3iotechnology 2; 627-659 for an overview). Basically, cells of all plant species are suitable for transformation. Both monocot and dicotyledonous plants are of interest. Transformation techniques have already been described for various monocot and dicot plant species. Cells of agricultural crops are preferably used in the processes, in particular of cereals, for example rye, oats, barley, wheat, potatoes, maize, rice, rapeseed, peas, sugar beet, soybeans, tobacco, cotton, sunflower, oil palm, wine , Tomato etc. or cells of ornamental plants. The invention also relates to the transgenic plant cells obtainable from the process and plants obtainable therefrom by regeneration, which have an increase in the rate of glycolysis due to the additional expression of a cytosolic invertase and a cytosolic hexokinase.

Furthermore, the present invention relates to propagation material of plants according to the invention which contains cells according to the invention. This can be any type of tissue or organ of the plants according to the invention which enables the propagation. These include, for example, tissue cultures of cells according to the invention, seeds, fruits, rhizomes, cuttings, seedlings, tubers etc.

The present invention further relates to recombinant DNA molecules which contain a DNA sequence which codes a protein with the enzymatic activity of a hexokinase, preferably a glucokinase, in combination with DNA sequences which transcription and translation in ensure plant cells. The hexokinase is is preferably a deregulated or unregulated enzyme.

The present invention further relates to recombinant DNA molecules which comprise the following DNA sequences:

(i) a DNA sequence which encodes a cytosolic invertase, in combination with DNA sequences which ensure transcription and translation in plant cells; and

(ii) a DNA sequence which codes a cytosolic hexokinase in combination with DNA sequences which ensure transcription and translation in plant cells.

The same applies to the selection of the DNA sequences which allow transcription and translation in plant cells as for the recombinant DNA molecules, which has already been stated above in connection with the cells and methods according to the invention, as well as the choice of the DNA sequences which code for an invertase or hexokinase.

Finally, the present invention relates to the use of DNA sequences which encode an invertase, preferably a deregulated or unregulated, for the production of transgenic plant cells which have an increased glycolyzerate in comparison to non-transformed plant cells. The invention also relates to the use of DNA sequences which encode a hexokinase, preferably a deregulated or unregulated, for the production of transgenic plant cells which have an increased glycolytic rate in comparison to non-transformed plant cells. Description of the picture

Figure 1 shows the 12.68 kb plasmid pB33Hyg-GK. The plasmid contains the following fragments:

A = Fragment A (1498 bp) contains the Dral-Dral fragment (position -1512 to position +14) of the promoter region of the patatin gene 333 (Rocha-Sosa et al., EMBO J. 8 (1989), 23-29).

B = fragment B (1025 bp) contains a DNA fragment with the coding region of the Zymomonas mobilis glucokinase (GenEMBL accession number: M60615; nucleotides 5128 to 6153).

C = fragment C ^' .192 bp) contains the polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid pTi-ACH5, nucleotides 11749-11939.

Methods

1. Cloning procedure

The vector pUC18 was used for cioning in E. coli. For the plant transformation, the gene constructions were cloned into the binary vector pBinAR (Höfgen and Willmitzer, Plant Sei. 66 (1990), 221-233).

2. Strains of bacteria

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

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

4. Transformation of potatoes

Ten small leaves of a potato sterile culture (Solanum tuberosum L. cv. Desiree) 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 tumefaciens overnight culture grown under selection. After gently shaking for 3-5 minutes, another incubation was carried out for 2 days in the dark. Thereupon the leaves for callus induction on MS medium with 1.6% glucose, 5 mg / 1 naphthylacetic acid, 0.2 mg / 1 3enzylaminopurin, 250 mg / 1 claforan, 50 mg / 1 kanamycin 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, 50 mg / 1 kanamycin and 0.80% Bacto agar.

5. Radioactive labeling of DNA fragments

The radioactive labeling of DNA fragments was carried out with the aid of a DNA random primer labeling kit from Boehringer (Germany) according to the manufacturer's instructions. 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%.

7. Determination of the starch content and dry matter of potato tubers

The starch content and the dry substance of the potato tuber were determined by means of the determination of the specific weight (Scheele et al., Landw. Vers. Sta. 127 (1937), 67-96) according to the following formulas:

Dry = 24.182 + 211.04 x (special weight - 1.0988)

substance

Strength = 17.546 + 199.07 x (special weight - 1.0988)

Determination of phosphorylated metabolic intermediates in potato tubers

Phosphorylated metabolic intermediates in potato tubers were determined with minor changes according to Weiner and Stitt (Biochim. Biophys. Acta 893 (1987), 13-21).

Presentation of the potato tuber extract: Approx. 200 mg tuber material were homogenized in a mortar under liquid nitrogen. The phosphorylated intermediates were extracted with 16% trichloroacetic acid solution in diethyl ether. After three hours of incubation on ice, the trichloroacetic acid was removed by extracting three times with diethyl ether. The extracts were then neutralized with 5 M KOH / l M triethanolamine. The determination of Phosphoenolpyruvate (PEP) and pyruvate took place immediately. For the determination of the other intermediates, the extract can be kept at -70 ° C. for several days.

The phosphoryiated intermediates were determined on a two-wavelength photometer (Sigma ZWS 11) by means of coupled enzymatic reactions according to Stitt et al. (Methods in Enzymology 174, 518-552).

a) Determination of PEP and pyruvate

The reaction buffer contained: 50 mM Hepes-KOH pH 7.0;

5 mM MgCl ₂ ;

0.025 mM NADH;

1 mM ATP pyruvate determination: 1 U / ml lactate dehydrogenase PEP determination: +2 U / ml pyruvate kinase

The measurement is carried out at 25 ° C. with 50 to 100 μl extract.

b) Determination of UPD glucose (UDPG)

The reaction buffer contained: 200 mM glycine pH 8.7;

5 mM MgCl ₂ ;

1mM NAD; 0.025 U / ml UDP-glucose dehydrogenase The measurement is carried out at 25 ° C. with 50 to 100 μl extract.

c) Determination of glucose-6-phosphate (G6P), Fmctose-6-phosphate (F5P) and glucose-1-phosphate (G1P)

The reaction buffer contained: 50 mM Hepes-KOH pH 7.0;

5 mM MgCl ₂ ;

0.25 mM NADP G6P determination: 2 U / ml glucose-6-phosphate dehydrogenase FlP determination: + 2 U / ml phosphoglucoisomerase GlP determination: + 2 U / ml phosphoglucomutase The measurement is carried out at 25 ° C with 50 to 100 μl extract. d) Determination of ATP

The reaction buffer contained: 50 mM Hepes-KOH pH 7.0;

5 mM MgCl ₂ ; 0.25 mM NADP; 1 mM glucose; 2 U / ml glucose-6-phosphate dehydrogenase; 2 U / ml phosphoglucoisomerase; 1 U / ml hexokinase The measurement is carried out at 25 ° C. with 50 to 100 μl extract

e) Determined by ADP

The reaction buffer contained: 50 mM Hepes-KOH pH 7.0;

5 mM MgCl ₂ ; 0.05 mM NADH; 0.2 mM PEP; 0.2 U / ml lactate dehydrogenase; 1 U / ml pyruvate kinase The measurement is carried out at 25 ° C. with 50 to 100 μl extract f) determination of 3-phosphogluccerate (3-PGA)

The reaction buffer contained: 100 mM Tris-HCl pH 8.1;

5 mM MgCl ₂ ; 0.05 mM NADH, 1.33 mM ATP; 0.1 U / ml phosphoglycerate kinase; 2 U / ml glyceraldehyde phosphate dehydrogenase The measurement is carried out at 25 ° C. with 50 to 100 μl extract

9. Determination of the activity of enzymes of carbohydrate metabolism in potato tubers

For the determination of enzyme activities (e.g. glucokinase, fructokinase, sucrose synthase, invertase, phosphoglucomutase, phosphofructokinase, glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase or pvruvatkinase) in potato tubers, 200 mg of Knoilen material in 500 μl extraction buffer (50 mM Hepes-KOH pH 7.5; 5 mM MgCl ₂ ; 1 mM EDTA; 1 mM EGTA; 1 mM DTT; 2 mM benzamidine, 2 mM e-amino-n-caproic acid; 0.5 mM PMSF; 10% (v / v) glycerin; 0.1% (v / v) triton X- 100) homogenized. After centrifugation, 10 to 40 μl of the cell-free desalted extract are used for the enzyme activity measurement. With the exception of the sucrose synthase and invertase activity, the enzyme activities were determined by means of coupled enzymatic reactions on a spectrophotometer.

Glucokinase and fructokinase activity was determined according to Renz et al. (Planta 190 (1993), 156-165), the sucrose synthase and invertase activity according to Zrenner et al. (Plant J. 7 (1995), 97-107), the phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, pvruvate kinase activity according to Burell et al. (Planta 194 (1994), 95-101), and the phosphoglucomutase activity according to Pressey (Journal of Food Science 32 (1967), 381-385).

10. Determination of the gas exchange of potato tubers

Tubers (approximately 30 g) were taken directly from plants from the greenhouse and placed in an infrared gas analyzer (Binos 100 Rosemound, Dusseldorf, FRG) within 30 minutes. The CO ₂ production was determined over 20 minutes at 20 ° C. and evaluated with the software from Waltz (Effeltrich, FRG).

The examples illustrate the invention. example 1

Construction of the binary plasmid p33-Cy-Inv

The preparation of the plasmid p33-Cy-Inv and introduction of the plasmid into the genome of the potato has been described in EP-A2 0 442 592. Regenerated potato plants that were transformed with the binary construct p33-Cy-Inv were transferred into soil and selected for the invertase activity. Several genotypes were identified which have an invertase activity which is up to a hundred times higher than that of the control plants.

Example 2

Construction of the binary plasmid pB33Hyg-GK

For the plant transformation, the coding region of the glucokinase gene from Zymononas mobilis was amplified using the polymerase chain reaction (PCR) starting from genomic Zymomonas mobilis DNA. The sequence of the Zymomonas mobilis glucokinase is entered in the GenEMBL database with the Accessicn number M60615. The amplified fragment corresponds to the region from nucleic acids 5128 to 6153 of this sequence. An Asp718 interface was inserted at the 5 'end and a HindIII interface at the 3' end. The 1025 bp PCR fragment was cloned into the vector pUCBM20 via the two additional interfaces. Starting from this plasmid, the entire coding region of the glucokinase was subcloned into the vector pBluescriptSK after restriction digestion with EcoRI and HindIII. In extracts from Ξ. coli cells containing the resulting plasmid pSK-GK were found to have a 100-fold increase in glucokinase activity compared to extracts from untransformed E. coli cells. For this purpose, the cells of a 20 ml overnight culture were harvested and resuspended in 500 μl extraction buffer (30 mM KH ₂ PO ₄ ; 2 mM MgCl ₂ ; 10 mM 2-mercaptoetha- nol; 0.1% (Vol. / Vol.) Nonidεt? 40). After adding the same volume of acid-washed glass beads (0.1 mm diameter), the suspension was mixed vigorously four times for 30 seconds. After centrifugation, the glucokinase activity in the cell-free extract was carried out as in Scopes et al. (Biochem. J. 228 (1985), 627-634).

After the functionality of the PCR product had been verified in this way, the insert was recloned into a binary vector derived from pBIN19 (Bevan, Nucl. Acids Res. 12 (1984), 8711-8720). The following plasmid was created: the plasmid pB33Hyg-GK (cf. FIG. 1). As a promoter for the expression of a transgene in plants, the construct contains the B33 promoter from Solanum tuberosum (Rocha-Sosa et al., ΞMBO J. 8 (1989), 23-29). The construct pB33Hyg-GK was created as follows: Since the construct was to be used for the transformation of already transgenic potato plants which express the NPT-II gene, the plasmid pBIB, which codes for the HPT gene, was used which contains hygromycin B phosphotransferase (Becker, Nucl. Acids Res. 18 (1990), 203). The promoter of the B33 gene from Solanum tuberosum was inserted as a Dral fragment (position -1512 to +14 according to Rocha-Sosa et al., EMBO J. 8 (1989), 23-29) into the Sacl Cloned the plasmid pUC19. As an EcoRI / Smal fragment, the promoter region was cloned into the binary vector pBIN19, which contains the termination signal of the octopine synthase gene from Agrobacterium tumefaciens in the direct vicinity of a polylinker from M13mpl9. This resulted in pB33. The promoter polylinker terminator fragment of plasmid pB33 was cloned as an EcoRI / HindIII fragment into the plasmid pBIB linearized with EcoRI and HindIII. This resulted in the plasmid pB33Hyg.

The coding region of the glucokinase was then isolated after Asp718 / Sall digestion of the plasmid pSK-GK and Asp718 / Sall was cloned into the plasmid pB33Hyg. This resulted in the plasmid pB33Hyg-GK, which is used for the transforma- tion of the transgenic potato line U-Inv-2 (line 30) was used.

To transform Agrobacterium tumefaciens, the binary plasmid is introduced into the cells by direct transformation according to the method of Höfgen & Willmitzer (Nucl. Acids Res. 16 (1988), 9877). The plasmid DNA of transformed agrobacteria was determined by the method of Birnboim et al. (Nucl. Acids Res. 7 (1979), 1513-1523) isolated and analyzed by gel electrophoresis after suitable restriction cleavage. For the transformation of potato plants, for example, 10 small leaves of a sterile culture, wounded with a scalpel, are placed in 10 ml of MS medium with 2% (w / v) sucrose, which contains 30 to 50 μl of Agrobacterium tumefaciens grown under selection - Contains overnight culture. After 3 to 5 minutes of gentle shaking, the Petri dishes are incubated at 25 ° C in the dark. After 2 days, the leaves are placed on MS medium with 1.6% (w / v) glucose, 2 mg / 1 zeatin ribose, 0.02 mg / 1 naphthylacetic acid, 0.02 mg / 1 giberellic acid, 500 mg / 1 Claforan, 3 mg / 1 hygromycin and 0.8% Bacto agar. After a week's incubation at 25 ° C. and 3000 lux, the claforane concentration in the medium is reduced by half. The further cultivation was carried out as described by Rocha-Sosa et al. (EMBO J. 8 (1989), 23-29).

Example 3

Analysis of transgenic potato plants containing an invertase from yeast and a glucokinase from Zymomonas mobilis in

Express tubers

Regenerated potato plants of the line U-Inv-2 (30), which had been transformed with the binary construct pB33Hyg-GK, were transferred into soil and selected for the glucokinase activity in tubers. Several genotypes were identified, which showed up to five times increased glucokinase activity compared to the control plants, have (e.g. GK-41, GK-29, GK-38). It was also demonstrated that these genotypes continue to express the yeast invertase gene (see Table I).

Table I

Plant invertase activity glucokinase activity

(nmol min ^" mg ^" protein) (nmol min ^" mg ^" protein)

Control 9 + 1 11 + 2

U-Inv-2 1027 ± 44 18 + 2

GK-41 n.d. 43 ± 5

GK-29 n.d. 45 ± 5

GK-33 1132 ± 232 55 ± 13

The enzyme activities shown here are the average of at least five measurements based on five independent plants.

The above-mentioned genotypes GK-41, GK-29 and GK-38 were amplified and 15 plants in each case were transferred to a greenhouse. The tubers were harvested 4 months later.

Surprisingly, it was found that the tubers of the transgenic plants GK-41, GK-29 and GK-38 only contain 40 to 60% of the starch in control plants, but the starch-free proportion of dry matter remains constant (cf. Table II). This shows that only the starch content in the tubers of the GK plants is reduced by the expression of the invertase in combination with the expression of the glucokinase. A depression in yield of 25% was also found. This in turn correlates with the reduction in the starch content. Table II

Plant yield per specific% starch% dry

Plant weight substance

Control 191 g 1.091 16.1 22.5

U-Inv-2 147 g 1.074 12.7 18.9

GK-41 139 g 1.064 10.7 16.8

GK-29 135 g 1.058 9.5 15.5

GK-41 137 g 1.046 7.1 13.4

The analysis of soluble sugars such as glucose, fructose and sucrose surprisingly showed that the seven-fold increase in the glucose concentration in the U-Inv-2 plants compared with the wild type is greatly reduced by the expression of the glucokinase, so that the amount of glucose only is still 30% of the amount of glucose in tubers of WT control plants. The fructose concentration in the transgenic lines has not changed compared to the control plants. The strong reduction in the amount of sucrose in the U-Inv-2 plants is partially offset by the expression of the glucokinase (cf. Table III). In summary, it was found that e.g. the tubers of the GK-38 plants only contain 40% starch and 50% soluble sugar compared to tubers from untransformed control plants.

Table III

Plant glucose fructose sucrose in μmol g ^"1 fresh weight

Control 3.5 ± 2.2 0.9 ± 0.2 12.0 ± 1.0 U-Inv-2 25.7 ± 3.1 0.6 ± 0.3 0.7 ± 0.4 GK- 38 1.0 ± 0.7 0.2 ± 0.1 6.3 + 1.4

Since there is no evidence whatsoever that the amount of photoassociates which is transported from the mature leaves into the tubers via the phloem is reduced in the GK plants, on the other hand, however, reduced levels of starch and soluble sugars are measured, it can be assumed that the expression of an invertase in the cytosol of plant cells in combination with the expression of a glucokinase in cytosol of plant cells into one. Increases glycolysis and respiration results.

This surprising result is underlined by changed levels of metabolites and changed enzyme activities in tuber extracts of the GK plants. There was an up to five-fold increase in the glucose-6-phosphate content, an up to five-fold increase in the fructose-6-phosphate content, an approximately 40% increase in the 3-phosphoglycerate content, an approximately six-fold increase in the pyruvate content and an up to 50% Increase in the ATP content detected (see Table IV).

Table IV

Metabolite control U-Inv-2 GK-38

Glucose-6-phosphate 107 ± 15 343 ± 19 513 ± 56

Glucose-1-phosphate 13 ± 1 25 ± 2 17 ± 4

Fructose-6-phosphate 29 ± 5 100 ± 6 153 ± 19

UDP-glucose 126 ± 12 91 + 11 107 ± 4

3-phosphoglycerate 92 ± 18 127 ± 22 135 ± 35

Phosphoenolpyruvate 33 ± 4 34 ± 8 37 ± 11

Pyruvate 15 ± 3 27 ± 5 84 ± 23

ATP 32 ± 2 27 ± 7 45 ± 7

ADP 24 ± 2 25 ± 4 28 ± 2

The metabolite amounts shown here are the average of at least five measurements based on five independent plants. The values are given in nmol g ^"1 fresh weight.

Furthermore, it was shown that the activity of enzymes which catalyze glycolysis reactions is increased in tuber extracts from the GK plants (cf. Table V). For example, the specific activities of fructokinase, phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase (GAP-DH) and pvruvate kinase are increased. The expression of an invertase in the cytosol of plant cells in combination with the expression of a glucokinase in the cytosol of plant cells thus represents a method which leads to an increase in glycolysis and respiration.

Table V

Enzyme control U-Inv-2 GK-38

Sucrose synthase 41 + 5 15 + 4 236 ± 85

Fructokinase 52 + 2 90 + 7 88 ± 5

Phosphoglucomutase 1635 ± 150 1354 ± 79 1408 + 24

54 + 4 89 + 7 98 + 4

GAP-DH 942 ± 56 1791 ± 190 1926 + 145

Phosphoglycεratkir.ase 883 + 83 962 ± 78 901 + 22

Phosphoglyceratir.utase 539 + 68 553 ± 19 615 ± 21

Pyruvate kinase 532 ± 42 551 ± 29 688 ± 33

The enzyme activities shown here are the average of at least five measurements based on five independent plants. The values are given in nmol min ^" mg fresh weight.

Example 4

Determination of the gas exchange of tubers

The CO ₂ production was determined in the case of tubers which were still growing (Table VI). Table VI

Control U-Inv-2 GK-38

C0 ₂ 18 ± 2.0 58 ± 7.0 84 ± 6.0

The values shown here are the mean values of six measurements based on six independent plants. The values are given in mmol C0 ₂ g fresh weight.

The data given in Table VI show that the production of carbon dioxide is increased by a factor of 3 in the U-Inv-2 plants and by a factor of 5 in the GK plants. This surprising result means that the expression of an invertase in the cytosol in combination with the expression of a glucokinase is a process which leads to an increase in glycolysis and respiration in plant cells.

The controls given in the experiments described above are each non-transformed plants of the plant species or subspecies used for the transformation.

Claims

Patent claims

1. Transgenic plant cell with an increased glycolysis in comparison to non-transformed plant cells, in which it is due to the introduction and expression of DNA sequences which encode a cytosolic invertase, as well as of DNA sequences which contain a cytosoli ¬ encode hexokinase, there is an increase in invertase and hexokinase activity.

2. Transgenic plant cell according to claim 1, wherein the invertase is a deregulated enzyme.

3. Transgenic plant cell according to claim 1, wherein the invertase is an unregulated enzyme.

4. Transgenic plant cell according to one of claims 1 to 3, wherein the hexokinase is a deregulated enzyme.

5. Transgenic plant cell according to one of claims 1 to 3, wherein the hexokinase is an unregulated enzyme.

6. Transgenic plants according to one of claims 1 to 5, wherein the hexokinase is a glucokinase.

7. Transgenic plant cell according to one of claims 1 to 6, wherein the DNA sequence encoding the invertase encodes an invertase of a fungus.

8. The transgenic plant cell according to claim 7, wherein the invertase is an invertase from Saccharomyces cerevisiae.

9. The transgenic plant cell of claim 8, wherein the DNA sequence encoding the invertase is the Suc2 gene from Saccharomyces cerevisiae.

10. Transgenic plant cell according to one of claims 1 to 9, wherein the hexokinase is a hexokinase from a proka¬ ryontischen organism.

11. The transgenic plant cell according to claim 10, wherein the hexokinase is a glucokinase from Zymomonas mobilis.

12.. Transgenic plant cells according to one of claims 1 to

11, the DNA sequence encoding the invertase being under the control of a tissue-specific promoter, a promoter which is active at a specific development time of the plant or a promoter which can be induced by external factors.

13. Transgenic plant cell according to one of claims 1 to

12, the DNA sequence encoding the hexokinase being under the control of a tissue-specific promoter, a promoter which is active at a specific developmental time in the plant, or a promoter which can be induced by external factors.

14. Transgenic plant cell according to one of claims 1 to

13, which is a cell of a crop.

15. A transgenic plant cell according to claim 14, which is a cell of an oil-storing plant.

16. Transgenic plant cell according to claim 15, which is a cell of a rapeseed, soybean, sunflower or oil palm plant.

17. The transgenic plant cell according to claim 14, which is a cell of a starch-storing plant.

18. Transgenic plant cell according to claim 17, which is a cell of a maize, rice, wheat, barley, rye, oat or potato plant.

19. Transgenic plants obtainable by regeneration of a plant cell according to one of claims 1 to 18.

20. Transgenic plant containing plant cells according to one of claims 1 to 18.

21. Transgεnε plant according to claim 19 or 20, in which the transport rate of photoassimilates is increased due to the increase in the rate of glycolysis in transfer cells.

22. Transgenic plant according to one of claims 19 to 21, in which the absorption of minerals from the soil is increased due to the increase in the rate of glycolysis in cells of the root epidermis or root hair.

23. Transgenic plant according to one of claims 19 to 22, in which the fatty acid bio-synthesis is increased due to the increased glycolysis rate in the endosperm and / or in the cotyledons of seeds.

24. The transgenic plant according to any one of claims 19 to 23, in which, due to the increased glycolysis rate in an organ, the fatty acid content is increased with a simultaneous reduction in the starch content.

25. Propagation material of a plant according to one of claims 19 to 24, containing transgenic plant cells according to one of claims 1 to 18.

26. A process for the production of transgenic plant cells with an increased glycolysis rate, in which DNA sequences which encode a cytosolic invertase are introduced into plant cells, and DNA sequences which encode a cytosolic hexokinase, and these DNA sequences are expressed in the transformed plant cells.

27. The method according to claim 26, wherein the invertase is a de-regulated or unregulated enzyme.

28. The method according to claim 26 or 27, wherein the hexokinase is a deregulated or unregulated enzyme.

29. Vεrfahrεn according to εinem of claims 26 to 28, wherein the hexokinase is a glucokinase.

30. Recombinant DNA molecule containing the following DNA sequences:

31. Recombinant DNA molecule containing a DNA sequence that encodes a protein with the enzymatic activity of a hexokinase, in combination with DNA sequences that ensure transcription and translation in plant cells.

32. Recombinant DNA molecule according to claim 30 or 31, wherein the hexokinase is a glucokinase.

33. Use of DNA sequences which encode an invertase for the production of transgenic plant cells which have an increased glycolysis rate in comparison with non-transformed plant cells.

34. Use of DNA sequences which encode a hexokinase for the production of transgenic plant cells, which have an increased glycolysis rate compared to non-transformed plant cells.

35. Use according to claim 3.3, wherein the hexokinase is a glucokinase.