AU719452B2 - Transgenic plant cells and plants with an increased glycolysis rate - Google Patents

Description

VOSSIUS PARTNER Gesellschaft burgerlichen Rechts SIEBERTSTRASSE 4 81675 MUNCHEN TELEFON +49-89-41304-0 FAX G 3: +49-89-41304-111 FAX G 4: +49-89-41304-101 Transgenic plant cells and plants with an increased glycolysis rate The present invention relates to transgenic plant cells and plants exhibiting an increased glycolysis rate when compared to non-transformed plants. The increase of the glycolysis rate results from introducing and expressing a DNA sequence encoding a cytosolic invertase, preferably a deregulated or unregulated invertase, as well as from introducing and expressing a DNA sequence encoding a cytosolic hexokinase, preferably a deregulated or unregulated hexokinase, into 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 encoding proteins with the enzymatic activity of an invertase or proteins with the enzymatic activity of a hexokinase in order to produce plants exhibiting an increased glycolysis rate.

Due to the continuously increasing demand of foodstuffs resulting from the ever-growing world population it is an object of biotechnological research to strive to increase the yield of useful plants. A possibility to achieve this aim is to specifically modify the metabolism of plants by means of recombinant DNA techniques. Targets for such modifications may for example be the primary processes of photosynthesis which lead to CO 2 fixation, the transport processes which take part in distributing the photoassimilates within the plant or also the metabolic pathways which lead to the synthesis of storage substances such as starch, proteins or oleic substances. It is described, for example, that the expression of a prokaryotic \asparagine synthetase in plant cells leads among other things to an increase of the biomass production in the transgenic plants (EP-B 0 511 979). It was also proposed to express a prokaryotic polyphosphate kinase in the cytosol of transgenic plants which in the case of potato plants leads to an increased yield of up to 30% with regard to the weight of the tubers.

Moreover, EP-A2 0 442 592 describes the expression of an apoplastic invertase in potato plants which also leads to an increased yield of transgenic plants transformed in such a way.

Further experiments were carried out with regard to the modification of the activity of enzymes involved in the synthesis of sucrose as one of the most important transport metabolites in most plants (cf. e.g. Sonnewald et al., Plant Cell and Environment 17 (1995), 649-658).

WO 91/19896 also describes that the increase of the starch biosynthesis by overexpressing a deregulated enzyme of the ADP glucose pyrophosphorylase leads to an increased allocation of sucrose into potato tubers, where it is stored in the form of starch.

Whereas many of these uses refer to the steps which either lead to the formation of photoassimilates in leafs (cf. also EP 466 995) or to the formation of polymers such as starch or fructanes in the storage organs of transgenic plants

WO

94/04692) up to now there have not been any promising approaches describing the modifications which have to be effected in the primary metabolic pathways in order to achieve an increase of the glycolysis rate. An increase of the glycolysis rate is significant, for instance, for all those processes in the plants for which considerable amounts of ATP are needed. This is true, for example, for a number of transport processes via membranes which are driven by a membrane potential or a proton gradient which is produced by the activity of a H'-ATPase. Moreover, an increase of the glycolysis rate is significant for the growth within the meristem, for the change from vegetative to generative growth as well as for the synthesis of various storing substances, especially oils.

Thus, the object of the present invention is to provide plant cells and plants with an increased glycolysis rate as well as methods and DNA molecules for their production.

This problem is solved by providing the embodiments described in the claims.

Therefore, the present invention relates to transgenic plant cells exhibiting an increased glycolysis rate when compared to non-transformed plant cells, which show, due to the introduction and expression of DNA sequences encoding a cytosolic invertase and of DNA sequences encoding a cytosolic hexokinase, an increase of the invertase and hexokinase activity. The transgenic cells may each contain one or more DNA sequences encoding an invertase or a hexokinase.

Surprisingly it was found that by introducing and expressing DNA sequences encoding a protein with the enzymatic activity of a hexokinase and by simultaneously introducing and expressing DNA sequences encoding a protein with the enzymatic activity of an invertase within the cytosol of plant cells, a drastic increase in the glycolysis rate of plant cells transformed in such a way when compared to non-transformed plant cells may be achieved. The expression of an invertase of fungal origin within the cytosol was already described (cf. EP-A2 442 592) However, disclosed was only the expression of fungal invertase within the cytosol of plant cells alone. The use of the cytosolic invertase in order to increase the glycolysis rate, particularly in combination with the expression of a hexokinase, was not described.

An increased glycolysis rate means that the transgenic plant cells which have been transformed with DNA sequences leading to the additional synthesis of an invertase and of a hexokinase within the cytosol of the cells exhibit an increased glycolysis rate when compared to non-transformed plant cells, preferably a glycolysis rate which is increased by at least 30%, more Veeferably one which is increased by at least 50% to 100% and particularly one which is increased by more than 100% to 200% and most preferably one which is increased 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 of the glycolysis rate may be determined, for example, by the increase in the concentration of glucose-6-phosphate, fructose-6-phosphate, 3phosphoglycerate, pyruvate or ATP. Within the framework of the present invention the increase of the glycolysis rate is preferably determined by assessing the increase of pyruvate.

In this context, the increase of the glycolysis rate is determined in comparison with non-transformed cells. That means that material of plants which have been used as a starting material for the introduction of the above-mentioned DNA sequences is now used for determining the glycolysis rate. The glycolysis rate determined in such a way is then compared to the glycolysis rate of plant material of a corresponding species or plant line after transformation with the abovedescribed DNA sequences.

The provision of an increased amount of ATP by means of an increased glycolysis rate enables, for example, the increase of various energy-depending 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 may be used, for examples, for the intensified synthesis of oils, fats or isoprenoid derivatives. If DNA sequences enabling the synthesis of polyhydroxyalkanoic acids are simultaneously expressed within the cells, acetylCoA may also be used for the formation of such alkanoic acids.

In a preferred embodiment of the invention the DNA sequences encoding an invertase or, as the case may be, a hexokinase are DNA sequences encoding an invertase or, as the case may be, a hexokinase which, when compared to invertases and hexokinases usually occurring in plants, are deregulated or unregulated. In is regard, deregulated means that these enzymes are not regulated in the same way as the invertase and hexokinase enzymes usually formed in non-modified plant cells. In particular, these enzymes are subject to different regulation mechanisms, i.e. they are not to the same extent inhibited by the inhibitors present in plant cells or allosterically regulated by metabolites. In this respect, deregulated preferably means that the enzymes exhibit a higher activity than invertases or hexokinases endogeneously expressed in plant cells. Within the framework of the present invention, unregulated means that the enzymes are not subject to any regulation in plant cells.

The DNA sequences encoding a protein with the enzymatic activity of an invertase may be DNA sequences encoding prokaryotic invertases, particularly bacterial invertases. They may also be DNA sequences encoding eukaryotic invertases, i.e.

invertases from plants, alga, fungi or animal organisms. Within the framework of the present invention the term fungi also refers to kinds of yeast, especially yeast of the genus Saccharomyces, such as Saccharomyces cerevisiae. The enzymes encoded by the sequences may be known and naturally occurring enzymes exhibiting a deviating regulation by various substances, in particular by plant invertase inhibitors. They may also be enzymes which were generated by mutagenesis of DNA sequences encoding known enzymes from bacteria, alga, fungi, animals or plants.

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, in comparison to plant invertases, they are not regulated by plant invertase inhibitors. Preferably, use is made of DNA sequences encoding -an invertase from Saccharomyces. Such sequences are known and were described (cf. Taussig et al., Nucleic Acids Res. 11 (1983), 1943-1954; EP A2 0 442 592). In order to ensure e localization of the invertase within the cytosol of the pnt cell, possibly occurring DNA sequences encoding signal peptides have to be deleted and the coding region has to be provided, if necessary, with a new start codon (cf. EP-A2 0 442 592) Apart from the above-mentioned DNA sequence from Saccharomyces cerevisiae other DNA sequences are known which encode 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 L. 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, X81792 C. rubrum, X81793 C. rubrum, X77264 L. esculentum, Z12027 L.

esculentum, D10465 Z. mobilis, D17524 Zymomonas mobilis) and which due to their properties can also be used in order to produce the plant cells of the invention. Care must be taken that the protein is formed within the cytosol of the plant cell. Methods for the modification of such DNA sequences in order to ensure the localization of the synthesized enzymes within the cytosol of the plant cell are known to the skilled person. In the case that the invertases contain sequences necessary for secretion or for a specific subcellular localization, e.g. for the localization in the extracellular space or within the vacuole, the respective DNA sequences have to be deleted.

The DNA sequences encoding a protein with the enzymatic activity of a hexokinase may be DNA sequences which encode prokaryotic invertase, particularly bacterial invertases, as well as DNA sequences which encode eukaryotic invertases, i.e.

invertases from plants, alga, fungi or animal organisms.

Hexokinases (EC 2.7.1.1) are enzymes catalyzing the following reaction: N hexose ATP hexose-phosphate

ADP

The hexokinases encoded by the DNA sequences may be known enzymes occurring in nature which exhibit a deviating regulation by various substances. They may also be enzymes generated by mutagenesis of DNA sequences encoding known enzymes from bacteria, alga, fungi, animals or plants. DNA sequences encoding enzymes with hexokinase activity are described for a wide range of organisms such as Saccharomyces cerevisiae, humans, rat and various microorganisms (for the DNA sequences, see: EMBL gene bank, accession numbers M92054, L04480, M65140, X61680, M14410, X66957, M75126, J05277, J03228, M68971, M86235, X63658).

In a preferred embodiment the DNA sequences encode glucokinases, in particular glucokinases subjected to a reduced allosteric regulation, for example, by glucose-6-phosphate.

Glucokinases (EC 2.7.1.2) are hexokinases with a high affinity for glucose catalyzing the following reaction: glucose ATP glucose-6-phosphate

ADP

In a preferred embodiment the DNA sequences encode a glucokinase from Zymomonas mobilis (Barnell et al., J.

Bacteriol. 172 (1990), 7227-7240; EMBL gene bank accession number M60615). Other glucokinases are described for humans and rat (for the DNA sequences, see: EMBL gene bank accession numbers M69051, M90299, J04218 and M25807).

Moreover, DNA sequences encoding an invertase or a hexokinase may be isolated from any desired organism with the help of the already known above-described DNA sequences. Methods for the isolation and identification of such DNA sequences are known to the person skilled in the art, such as hybridization with known sequences or by means of polymerase chain reaction using primers derived from known sequences.

The enzymes encoded by the identified DNA sequences are subsequently examined with respect to their enzyme activity and regulation. Methods for determining the invertase or hexokinase <ativities are known to the skilled person.

By introducing mutations and modifications according to methods known to the skilled person the proteins encoded by the DNA sequences may further be altered in their regulatory properties in order to obtain deregulated or unregulated enzymes.

For the expression in plant cells the DNA sequences encoding a cytosolic invertase or hexokinase may generally be placed under the control of any desired promoter functioning in plant cells.

The expression of the aforesaid DNA sequences may generally take place in any tissue of a plant regenerated from a transformed plant cell of the invention at any point of time.

However, it preferably takes place in such tissues in which an increased glycolysis rate is an advantage either for the growth of the plant, for the absorption and the transport of ions and metabolites or for the formation of the metabolites in the plant. Therefore, promoters which ensure a specific expression within a certain tissue at a certain point of time of the plant's development or in a certain organ of the plant, appear to be most suitable. Thus, promoters which are specifically active within the endosperm or within the cotyledons of forming seeds appear to be particularly suitable for increasing the fatty acid biosynthesis due to an increased acetlylCoA content in the seeds of oil-generating plants such as oilseed rape, soy bean, sunflower and oil palms. Such promoters are, the phaseolin promoter from Phaseolus vulgaris, the USP promoter from Vicia faba or the HMG promoter from wheat.

Moreover, the use of promoters ensuring a seed-specific expression is advantageous. In the case of starch-storing plants such as maize, wheat, barley or other cereals the glycolysis rate is thereby increased in the seeds and the formation of pyruvate and acetylCoA as well as the biosynthesis of fatty acids are intensified. This means that the flow of photoassimilates is changed and is directed to pyruvatedependent biosynthetic pathways such as the biosynthesis of fatty acids rather than to starch.

omoters which are active within the storage organs such as t s or roots, e.g. within the storage root of sugar beet or within the tuber of potato, are also preferably used. In this case, the expression of the DNA sequences encoding an invertase or hexokinase, leads to a change of the direction of biosynthetic pathways in such a way that less sugar or starch is formed and that more pyruvate and acetylCoA is formed due to an increased glycolysis rate.

Furthermore, the expression of the DNA sequences may be controlled by promoters which are specifically activated in the moment of the induction of flowering or which are active within tissues necessary for the induction of flowering. Use may also be made of promoters which are activated at a certain point of time which is solely controlled by external influences such as light, temperature, chemical substances (see, for example, WO 93/07279). Promoters of interest for the increase of the absorption rate of ions from the soil due to an increased

ATP

content are, such promoters which exhibit an expression specifically in root hair or in root epidermis. Promoters of interest for increasing the export rate of photo assimilates from the leaf are, for example, promoters which exhibit an expression specifically in companion cells. Such promoters are known the promoter of the rolC gene from Agrobacterium rhizogenes) Furthermore, the DNA sequences encoding an invertase or hexokinase may advantageously be linked not only to a promoter but also with DNA sequences ensuring a further increase of the transcription such as the so-called enhancer elements, or with DNA sequences from within the transcribed region which ensure a more efficient translation of the synthesized RNA into the respective protein (so-called translation enhancers). Such regions may be derived from viral genes or suitable plant genes or may be produced synthetically. They may be homologous or heterologous with respect to the used promoter. The DNA sequences encoding an invertase or hexokinase are advantageously linked to 3 '-non-translated DNA sequences ensuring the termination of the transcription and the polyadenylation of the transcript. Such sequences are known and have been described, e.g. that of the octopine synthase gene from Agrobacterium tumefaciens. These sequences may be exchanged in any desired way.

Preferably, the DNA sequences encoding an invertase or hexokinase are stably integrated into the genome of the plant cells of the invention.

The transgenic plant cells exhibiting an increased glycolysis rate due to the additional expression of a cytosolic invertase and a cytosolic hexokinase may generally be cells of any desired plant species. Of interest are cells of monocotyledonous as well as cells of dicotyledonous plant species, in particular cells of starch storing plants, oil storing plants or of agriculturally useful plants such as rye, oats, barley, wheat, potato, maize, rice, oilseed rape, pea, sugar beet, soy bean, tobacco, cotton, sunflower, oil palm, wine, tomato etc. or cells of ornamental plants.

Apart from an increased glycolysis rate, the plant cells of the invention differ from corresponding non-transformed plant cells in that they contain foreign DNA sequences stably integrated into the genome which encode a cytosolic invertase or, as the case may be, a cytosolic hexokinase. In this context, the term "foreign DNA sequence" means the following: these may be DNA sequences which are heterologous with regard to the transformed plant cell, i.e. they do not naturally occur within such a plant cell. In the case of DNA sequences naturally occurring within the transformed plant cells, "foreign" means that they are integrated at a location within the genome of the transformed plant cell where they do not naturally occur, i.e.

they have a new genomic environment. This may be verified, by means of a Southern Blot analysis. Moreover, the DNA molecules introduced into the plant cells are normally recombinant DNA molecules, i.e. molecules which are composed of various segments which do not occur naturally in this composition.

A further subject matter of the present invention are transgenic plants containing the transgenic plant cells of the invention. Such plants may be generated from the plant cells of the invention by, regeneration.

The provision of plant cells with an increased glycolysis rate enables the production of transgenic plants with modified advantageous properties. By increasing the glycolysis rate, for instance, specifically within the companion cells of transgenic plants the loading of the sieve-element-companion-cell-complex with sucrose by the sucrose-proton-cotransporter may be increased, which leads to an increase of the transport rate of photoassimilates. In the same way the absorption of anorganic ions such as phosphate, sulfate, nitrate and others from the soil via the root may be increased.

The specific increase of the glycolysis rate in root cells, in particular in root hair and epidermal cells may, due to an increased H' ATPase activity, lead to( an intensified secretion of protons into the soil. Such an acidification of the soil leads to the mobilization and therefore to an easier absorption of various minerals, such as phosphate, from the soil.

By increasing the glycolysis rate in oil storing tissues of plants, such as the endosperm or the cotyledons of seed or other oil storing organs, the flow of the photoassimilates which are imported into the seeds or organs can be directed to the formation of pyruvate and acetylCoA. This possibility is of particular interest. The provision of elevated amounts of these intermediates for the biosynthesis of triglycerides leads to an increased synthesis of oils and therefore to an increased yield. The provision of elevated amounts of acetylCoA is also significant for many other processes naturally occurring in plants, such as isoprenoid biosynthesis, but also for the formation of polymers such as polyhydroxyalkanoic acids (cf.

e.g. Poivier et al., Bio/Technology 13 (1995), 142-150).

By increasing the glycolysis rate in starch storing tissues, e.g. in the seeds of various kinds of cereals or in potato tubers, the flow of photoassimilates is directed from the starch biosynthesis to pyruvate-dependent biosynthetic pathways, such as the fatty acid biosynthesis. This results in a reduction of the amount of starch in the respective tissue and possibly in a simultaneous increase of the fatty acid biosynthesis.

Furthermore it is significant that the high acetylCoA concentrations generated by the increase of the glycolysis rate may also lead to an intensified isoprenoid synthesis or, in combination with the expression of the corresponding genes for the synthesis of polyhydroxyalkanoic acids, to a polyhydroxyalkanoic acid synthesis within the transgenic plant cells.

Furthermore, the present invention relates to a method for the production of transgenic plant cells exhibiting an increased glycolysis rate when compared to non-transformed plant cells.

In this method, DNA sequences encoding a cytosolic invertase and DNA sequences encoding a cytosolic hexokinase are introduced into plant cells. These DNA sequences are then expressed in the transformed plant cells.

Such a method preferably comprises of the following steps: Production of a recombinant double-stranded DNA molecule comprising the following DNA sequences: a promoter ensuring the transcription in plant cells; (ii) a DNA sequence which encodes a cytosolic protein with the enzymatic activity of an invertase and which is linked to the promoter in sense-orientation; the production of a recombinant double-stranded

DNA

molecule comprising the following DNA sequences: a promoter ensuring the transcription in plant cells; (ii) a DNA sequence which encodes a cytosolic protein with the enzymatic activity of a hexokinase and is linked with the promoter in sense-orientation; and transfer of the DNA molecules produced according to step and into plant cells.

For the choice of the plant species, the promoters, further flanking DNA sequences, as well as for the choice and modifications of the DNA sequences encoding an invertase or, as the case may be, a hexokinase, the same holds true what has been mentioned above in connection with the cells of the invention.

The DNA sequences encoding an invertase or a hexokinase may either be localized on separated DNA molecules or together on one recombinant DNA molecule. If the sequences are situated on two different DNA molecules the transfer of the DNA molecules may either take place simultaneously or in such a way that the plant cells are first transformed with one DNA molecule and selected plant cells and plants are subsequently transformed with the second DNA molecule. Moreover, plants expressing an additional cytosolic invertase as well as an additional cytosolic hexokinase may be produced by initially producing two independent transgenic plant lines encoding an invertase or, as the case may be, a hexokinase, and by subsequently crossing the two plant lines.

The transfer of DNA molecules containing DNA sequences encoding the invertase or the hexokinase is preferably carried out by means of plasmids, particularly such plasmids which ensure a stable integration of the DNA molecule into the genome of transformed plant cells; examples for such plasmids are binary plasmids or Ti-plasmids of the Agrobacterium tumefaciens system. Apart from the Agrobacterium system other systems may be used for the introduction of DNA molecules into plant cells, such as the so-called biolistic method or the transformation of protoplasts (cf. for a review Willmitzer L. (1993), Transgenic Plants, Biotechnology 2, 627-659). Basically, cells of all plant species may be used for transformation. Monocotyledonous and dicotyledonous plants are particularly interesting. For various monocotyledonous and dicotyledonous plant species transformation techniques have been described. The cells of agriculturally useful plants are preferably used, e.g. rye, oats, barley, wheat, potato, maize, rice, oilseed rape, pea, sugar beet, soy bean, tobacco, cotton, sunflower, oil palm, wine, tomato etc. or cells from ornamental plants. The transgenic plants cells obtained from the described method and the plants regenerated therefrom, which due to the additional expression of a cytosolic invertase and a cytosolic hexokinase exhibit an increase of the glycolysis rate, are also the subject matter of the invention.

Moreover, the present invention relates to propagation material of plants of the invention, which contains the cells of the invention. The propagation material may be any kind of tissues or organs of the plants of the invention enabling propagation.

Among these are; for example, tissue cultures of the cells of the invention, seeds, fruits, rootstocks, cuttings, seedlings, tubers etc.

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

Furthermore, the present invention relates to recombinant DNA molecules comprising the following DNA sequences: a DNA sequence encoding a cytosolic invertase, in combination with DNA sequences ensuring the transcription and translation in plant cells; and (ii) a DNA sequence encoding a cytosolic hexokinase, in combination with DNA sequences ensuring the transcription and translation in plant cells.

For the choice of DNA sequences allowing for the transcription and translation in plant cells, and for the choice of the DNA \sequences encoding an invertase or, as the case may be, a hexokinase the same applies for recombinant DNA molecules what has been mentioned in connection with the cells and methods of the invention.

Finally, the present invention relates to the use of DNA sequences encoding an invertase, preferably a deregulated or unregulated invertase, for the production of transgenic plant cells exhibiting an increased glycolysis rate when compared to non-transformed plant cells. The use of DNA sequences encoding a hexokinase, preferably a deregulated or unregulated hexokinase, for the production of transgenic plant cells exhibiting an increased glycolysis rate when compared to nontransformed plant cells, is also a subject matter of the present invention.

Description of the Figure Fig. 1 shows the plasmid pB33Hyg-GK having a size of 12.68 kb.

The plasmid contains the following fragments: A Fragment A (1498 bp) contains the Dral DraI fragment (position -1512 to position +14) of the promoter region of the patatin gene B33 (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 glucokinase from Zymomonas mobilis (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 nucleotides 11749-11939.

Methods 1. Cloning methods For cloning in E.coli use was made of the pUC18 vector.

For plant transformation the gene constructs were cloned into the binary vector pBinAR (Hifgen and Willmitzer, Plant Sci. 66 (1990), 221-233).

2. Bacterial strains For the pUC vectors and for the pBinAR constructs use was made of the E.coli strain DH5a (Bethesda Research Laboratories, Gaithersburgh, USA).

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

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

4. Transformation of potatoes Ten small leaves of a sterile potato culture (Solanum tuberosum L. cv. D6sir6e) injured by a scalpel were placed into 10 ml MS medium (Murashige Skoog, Physiol. Plant.

(1962), 473) with 2% sucrose. The medium contained p1 of a Agrobacterium tumefaciens overnight-culture grown under selection. After gently shaking it for 3-5 minutes, another incubation took place in darkness for two days.

The leaves were subsequently put on MS medium with 1,6% glucose, 5 mg/l naphtyle acetic acid, 0,2 mg/l benzylaminopurine, 250 mg/l claforan, 50 mg/l kanamycin and 0,80% Bacto Agar for callus induction. After a oneweek incubation at 25 0 C and 3000 lux the leaves were put on MS-medium with 1,6% glucose, 1,4 mg/l zeatine ribose, mg/l naphtyle acetic acid, 20 mg/1 giberellic acid, 250 mg/l claforan, 50 mg/l kanamycin and 0,80% Bacto Agar for shoot induction.

Radioactive labelling of DNA fragments The radioactive labelling of DNA fragments was carried out by means of a DNA-Random Primer Labelling Kits by Boehringer (Germany) according to the manufacturer's instructions.

6. Plant maintenance Potato plants were kept in the greenhouse under the following conditions: light period 16 hours at 25000 lux and 22°C dark period 8 hours at 15 0

C

atmospheric humidity 7. Determination of the starch content and the dry matter of potato tubers The determination of the starch content and of the dry matter of the potato tuber was carried out by means of the determination of the specific weight (Sch6ele et al., Landw. Vers. Sta. 127 (1937), 67-96) according to the following formulae: dry 24.182 211.04 x (spec. weight 1.0988) matter starch 17.546 199.07 x (spec. weight 1.0988) 8. Determination of phosphorylated metabolic intermediates in potato tubers The determination of phosphorylated metabolic intermediates in potato tubers was carried out according to Weiner and Stitt (Biochim. Biophys. Acta 893 (1987), 13-21) with minor changes.

Preparation of the potato tuber extract: 200 mg of tuber material were homogenized under liquid nitrogen in a mortar. The phosphorylated intermediates were extracted with 16% trichloro acetic acid solution in diethylether. After a three-hours incubation on ice the trichloro acetic acid was removed by extraction with diethylether for three times. Afterwards the extracts were neutralized with 5 M KOH/1 M triethanolamine. The determination of phosphoenol pyruvate (PEP) and pyruvate was carried out immediately. For the determination of the other intermediates the extract may be stored at -70% for several days.

The determination of phosphorylated intermediates was carried out at a two-wavelengths 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 pyruvates The reaction buffer contained: 50 mM Hepes-KOH pH mM MgC1 2 0.025 mM NADH; 1 mM ATP Pyruvate determination: 1 U/ml lactate dehydrogenase PEP determination: +2 U/ml pyruvate kinase The measurement was carried out at 25 0 C with 50 to 100 p1 of extract.

b) Determination of UPD glucose (UDPG) The reaction buffer contained: 200 mM glycine pH 8.7; mM MgCl 2 1 mM NAD; 0.025 U/ml UDP glucose dehydrogenase The measurement was carried out at 25°C with 50 to 100 pl of extract.

c) Determination of glucose-6-phosphate (G6P), fructose- 6-phosphate (F6P) and glucose-l-phosphate (G1P) The reaction buffer contained: 50 mM Hepes-KOH pH mM MgC1 2 0.25 mM NADP G6P determination: 2 U/ml glucose-6-phosphate dehydrogenase F1P determination: 2 U/ml phosphoglucoisomerase G1P determination: 2 U/ml phosphoglucomutase The measurement was carried out at 25 0 C with 50 to 100 pl of extract.

d) Determination of ATP The reaction buffer contained: 50 mM Hepes-KOH pH mM MgC1 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 was carried out at 25°C with 50 to 100 pl of extract.

e) Determination of ADP The reaction buffer contained: 50 mM Hepes-KOH pH mM MgCl 2 0.05 mM NADH; 0.2 mM PEP; 0.2 U/ml lactate dehydrogenase; 1 U/ml pyruvate kinase The measurement was carried out at 250C with 50 to 100 ul of extract.

f) Determination of 3-phosphoglycerate (3-PGA) The reaction buffer contained: 100 mM Tris-HC1 pH 8.1; mM MgC1 2 0.05 mM NADH; 1.33 mM ATP; 0.1 U/ml phosphoglycerate kinase; 2 U/ml glyceraldehyde phosphate dehydrogenase The measurement was carried out at 25°C with 50 to 100 pl of extract.

9. Determination of the activity of enzymes of the carbohydrate metabolism in potato tubers In order to determine the enzyme activities of the glucokinase, fructokinase, sucrose synthase, invertase, phosphoglucomutase, phosphofructokinase, glyceraldehyde-3phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase or pyruvate kinase) in potato tubers, 200 mg of tuber material were homogenized in 500 pl 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-capronic acid; 0.5 mM PMSF; 10 (vol./vol.) glycerol; 0.1 (vol./vol.) Triton X-100).

After centrifugation 10 to 40 ul of the cell-free desalinated extract were used for the measurement of the enzyme activity. The enzyme activities, except for the sucrose synthase and invertase activity, were determined by means of a coupled enzymatic reaction in a spectral photometer.

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

Determination of the gas exchange of potato tubers Tubers (approx. 30 g) were taken directly from greenhouse plants and placed into an infra-red gas analyzer (Binos 100 Rosemound, Dusseldorf, FRG) within 30 minutes. The determination of the CO 2 production was carried out at 21 22 0 C for 20 minutes. Use was made of Waltz software (Effeltrich, FRG) for analysing the data.

For the purposes of this specification it will be clearly understood that the word "comprising" means "including but not limited to", and that the word "comprises" has a corresponding meaning.

The examples illustrate the invention.

Example 1 The construction of the binary plasmid p33-Cy-Inv The construction of the plasmid p33-Cy-Inv and the introduction of the plasmid into the potato genome has been described in EP-A2 0 442 592. Regenerated potato plants which had been transformed with the binary construct p33- Cy-Inv were transferred to soil and selected with regard to the invertase activity. Thereby, several genotypes were identified which exhibited a up to hundred times higher invertase activity than control plants.

o Example 2 The construction of the binary plasmid pB33Hyg-GK For plant transformation the coding region of the glucokinase gene from Zymomonas mobilis was amplified by means of the polymerase chain reaction (PCR), starting from genomic Zymomonas mobilis DNA. The sequence of the glucokinase from Zymomonas mobilis is deposited at the GenEMBL bank and has the accession number M60615. The amplified fragment corresponds to the region of the H:\Ninaw\Keep\Speci\68204-96.doc 10/12/99 22a nucleotides 5128 to 6153 of this sequence. An Asp7l8 restriction site was introduced at the 5'-end and a Hindill restriction side was introduced at the 3'-end. The PCR fragment with a length of 1025 bp was cloned over the two additional sites into the vector pUCBM2O vector. Using this plasmid, the complete codinig region of the glucokinase was U:\Ninaw\Keep\Speci\68204-96.doc 10/12/99 subcloned after cleavage with EcoRI and HindIII into the pBluescriptSK vector. In the extracts of E.coli cells containing the resulting plasmid pSK-GK a hundred-fold increase in glucokinase activity in comparison with extracts from untransformed E.coli cells was demonstrated. For this purpose, the cells of a 20 ml overnight culture were harvested and resuspended in 500 ul extraction buffer (30 mM KH 2

PO

4 2 mM MgCl 2 10 mM 2-mercaptoethanol; 0.1 (vol./vol.) Nonidet After the addition of the same volume of acid-washed glass pearls (0.1 mm in diameter) the suspension was heavily mixed four times for each 30 seconds. After centrifugation the glucokinase activity in the cell-free extract was assessed as described by Scopes et al. (Biochem. J. 228 (1985), 627-634).

After the functionality of the PCR product had thus been proven, the insertion was recloned into a binary vector derived from pBIN19 (Bevan, Nucl. Acids Res. 12 (1984), .8711-8720).

This led to the following plasmid: the plasmid pB33Hyg-GK (cf.

Fig. For the expression of a transgene in plants the construct contains the B33 promoter of Solanum tuberosum (Rocha-Sosa et al., EMBO J. 8 (1989), 23-29).

The construct pB33Hyg-GK was produced as follows: Since the construct was intended for the transformation of already transgenic potato plants expressing the NPT-II-gene, the plasmid pBIB, which contains the HPT gene coding for the hygromycin B phosphotransferase, was used (Becker, Nucl. Acids Res. 18 (1990), 203).

The promoter of the B33 gene of Solanum tuberosum was cloned as a Dral fragment (position -1512 to +14 according to Rocha-Sosa et al., EMBO J. 8 (1989), 23-29) into the SacI site of the pUC19 plasmid by means of polymerase II after degradation of the sticky ends. The promoter region was cloned into the binary vector pBIN19 as an EcoRI/Smal fragment. The pBIN19 vector contains the termination signal of the octopine synthase gene from Agrobacterium tumefaciens in direct neighborhood to a polylinker from M13mpl9. In this process, pB33 was formed. The promoter-polylinker-terminator fragment of the pB33 plasmid was cloned into the pBIB plasmid linearized with EcoRI and HindIII as an EcoRI/HindIII fragment. Thereby, the plasmid pB33Hyg was formed.

Subsequently, the coding region of the glucokinase was isolated from the plasmid pSK-GK after digestion with Asp718/SalI and was cloned as an Asp718/SalI fragment into the plasmid pB33Hyg.

This resulted in the formation of the plasmid pB33Hyg-GK which was used for the transformation of the transgenic potato line U-Inv-2 (line For the transformation of Agrobacterium tumefaciens the binary plasmid was introduced into the cells by means of direct transformation according to the method of HOfgen Willmitzer (Nucl. Acids Res. 16 (1988), 9877). The plasmid DNA of transformed Agrobacteria was isolated according to the method of Birnboim et al. (Nucl. Acids Res. 7 (1979), 1513-1523) and analyzed by means of gel electrophoresis after suitable restriction cleavage. In order to transform potato plants, for example, ten small leaves of a sterile culture injured by a scalpel were placed in 10 ml MS medium with 2% (weight/vol.) sucrose. The medium contained 50 pl of a Agrobacterium tumefaciens overnight-culture grown under selection. After gently shaking it for 3-5 minutes, the Petri dishes were incubated in darkness at 25'C. After two days the leaves were put on MS medium with 1.6% (weight/vol.) glucose, 2 mg/l zeatine ribose, 0.02 mg/l naphtyle acetic acid, 0.02 mg/l giberellic acid, 500 mg/l claforan, 3 mg/l hygromycine and 0.8 Bacto Agar. After a one-week incubation at 25 0 C and 3000 lux the claforan concentration in the medium was reduced by 50 A further cultivation was carried out according to Rocha-Sosa et al. (EMBO J. 8 (1989), 23-29).

Example 3 Analysis of transgenic potato plants expressing an invertase from yeast and a glucokinase from Zymomonas mobilis in tubers Regenerated potato plants of the U-Inv-2 line (30) which had been transformed with the binary construct pB33Hyg-GK were transferred into soil and selected with regard to the glucokinase activity in the tubers. Several genotypes were identified which exhibited an up to five times higher glucokinase activity than control plants GK-41, GK-29, GK-38). Furthermore, it was proved that these genotypes still express the invertase gene from yeast (cf. Table I).

Table I Plant Invertase activity Glucokinase activity (nmol min mg 1 protein) (nmol min' mg protein) Control 9 1 11 2 U-Inv-2 1027 44 18 2 GK-41 n.d. 43 GK-29 n.d. 45 GK-38 1132 232 55 13 The above-described enzyme activities are the average values of at least five measurements starting from five independent plants.

The above-mentioned genotypes GK-41, GK-29 and GK-38 were amplified and each 15 plants were transferred to a greenhouse.

The tubers were harvested after 4 months.

Surprisingly it was found that the tubers of the transgenic plants GK-41, GK-29 and GK-38 only contained 40 to 60 of the amount of starch of control plants whereby the starch-free amount of dry matter remains the same (cf. Table II) This nlhows that only the amount of starch in the tubers of GK-plants reduced due to the expression of the invertase in combination with the expression of the glucokinase.

Furthermore, it was found that the yield was repressed by 25 This correlates on the other hand with the reduction of the amount of starch.

Table II Plant Yield per Specific weight starch %dry plant matter 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 7-fold increase of the glucose concentration in the U-Inv-2 plants is strongly reduced due to the expression of glucokinase when compared to the wildtype. Thus, the amount of glucose is now only 30 of the amount of glucose in tubers of the WT control plants. The fructose concentration in the transgenic lines remains unaltered in comparison to the control plants. The strong reduction of the amount of sucrose in U-Inv-2 plants is partly neutralized by the expression of glucokinase (cf. Table III) In summary, it was found that e.g. the tubers of GK-38 plants contain only 40 of the starch and 50 of the soluble sugars that are contained in the tubers of the untransformed control plants.

Table III Plant Glucose Fructose Sucrose in pmol g-1 fresh weight Control 3.5 2.2 0.9 0.2 12.0 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 There is no indication that the amount of photoassimilates, which is transported from the mature leafs into the tubers via the phloem, is reduced within the GK-plants. On the other hand reduced percentages of starch and soluble sugars are measured.

Therefore, 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 the cytosol of plant cells leads to an increase of the glycolysis and the respiration.

This surprising result is emphasized by the altered contents of metabolites and the altered enzyme activities within the tuber extracts of the GK plants. It was found that the glucose-6phosphate content was increased up to five times, the fructose- 6-phosphate content was increased up to five times, the 3phosphoglycerate content was increased by approximately 40 the pyruvate content was increased approximately six times and the ATP content was increased by up to 50 (cf. 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 Phosphoenol pyruvate 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 above-shown amounts of metabolites are the average value of at least five measurements starting from five independent plants. The values are indicated in nmol g-1 fresh weight.

Furthermore, it was shown that the activity of enzymes which catalyze reactions of glycolysis is increased in the tuber extracts of the GK-plants (cf. Table V).

For example, the specific activities of fructokinase, phosphofructokinase, glyceraldehyde-3-phosphate-dehydrogenase (GAP-DH) and pyruvatekinase are increased.

The expression of an invertase within the cytosol of plant cells in combination with the expression of a glucokinase within the cytosol of plant cells thus constitutes a method leading to an increase of glycolysis and respiration.

Table V Enzyme Sucrose synthetase Fructokinase Phosphoglucomutase Phosphofructokinase

GAP-DH

Phosphoglycerate kinase Phosphoglycerate mutase Pyruvate kinase Control 41 5 52 2 1635 150 54 4 942 56 883 83 539 68 532 42 U-Inv-2 15 4 90 7 1354 79 89 7 1791 190 962 78 553 19 551 29 GK-38 236 88 1408 24 98 4 1926 145 901 22 615 21 688 33 The above-indicated enzyme activities are the average values of at least five measurements starting from five independent plants. The values are indicated in nmol min 1 mg 1 fresh weight.

Example 4 Determination of the gas exchange of tubers The CO 2 production was determined in still growing tubers (Table VI).

Table VI C0 2 Control 18 2.0 U-Inv-2 58 7.0 GK-38 84 The above-indicated values are the average values of six measurements starting from six independent plants. The values are indicated in nmol CO 2 g-1 fresh weight.

The data of Table VI show that the production of carbon dioxide is 3 times as high in U-Inv-2 plants and 5 times as high in GKplants.

This surprising result means that the expression of an invertase in the cytosol in combination with the expression of a glucokinase constitutes a method leading to an increase of the glycolysis and of the respiration in plant cells.

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

Claims (31)

1. A transgenic plant cell which shows, in comparison to non- transformed plant cells, an increased glycolysis rate and in which the invertase and hexokinase activity is increased due to the introduction and expression of DNA sequences encoding a cytosolic invertase as well as of DNA sequences encoding a cytosolic hexokinase.

2. The transgenic plant cell of claim 1, wherein the invertase is a deregulated enzyme.

3. The transgenic plant cell of claim 1, wherein the invertase is an unregulated enzyme.

4. The transgenic plant cell of any one of claims 1 to 3, wherein the hexokinase is a deregulated enzyme. The transgenic plant cell of any one of claims 1 to 3, wherein the hexokinase is an unregulated enzyme.

6. The transgenic plant of any one of claims 1 to 5, wherein the hexokinase is a glucokinase.

7. The transgenic plant cell of any one of claims 1 to 6, wherein the DNA sequence encoding the invertase encodes the invertase of a fungus.

8. The transgenic plant cell of 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. The transgenic plant cell of any one of claims 1 to 9, wherein the hexokinase is a hexokinase from a prokaryotic organism.

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

12. The transgenic plant cell of any one of claims 1 to 11, wherein the invertase-encoding DNA sequence is under the control of a tissue-specific promoter, of a promoter which is active in the plant at a certain developmental stage, or of a promoter which can be induced by external factors.

13. The transgenic plant cell of any one of claims 1 to 12, wherein the hexokinase-encoding DNA sequence is under the control of a tissue-specific promoter, of a promoter which is active in the plant at a certain developmental stage, or of a promoter which can be induced by external factors.

14. The transgenic plant cell of any one of claims 1 to 13 which is a cell from a useful plant. The transgenic plant cell of claim 14 which is a cell from an oil-storing plant.

16. The transgenic plant cell of claim 15 which is a cell from oilseed rape, soy bean, sunflower or oil palm.

17. The transgenic plant cell of claim 14 which is a cell from a starch-storing plant.

18. The transgenic plant cell of claim 17, which is a cell from maize, rice, wheat, barley, rye, oat or potato.

19. A transgenic plant obtainable by regeneration of a plant cell of any one of claims 1 to 18. i A transgenic plant containing plant cells of any one of claims 1 to 18.

21. The transgenic plant of claim 19 or 20, in which the transport rate of photoassimilates is increased due to the increase of the glycolysis rate in companion cells.

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

23. The transgenic plant of any one of claims 19 to 22, in which the biosynthesis of fatty acids is increased due to the increased glycolysis rate in the endosperm and/or in the cotyledons of seeds.

24. The transgenic plant of any one of claims 19 to 23, in which due to the increased glycolysis rate in an organ the fatty acid content is increased and at the same time the starch content is reduced. Propagation material of a plant of any one of claims 19 to 24 containing transgenic plant cells of any one of claims 1 to 18.

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

27. The method of claim 26, wherein the invertase is a deregulated or unregulated enzyme. 34

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

29. The method of any one of claims 26 to 28, wherein the hexokinase is a glucokinase. A recombinant DNA molecule comprising the following DNA sequences: a DNA sequence encoding a cytosolic invertase in combination with DNA sequences ensuring the transcription and translation in plant cells; and (ii) a DNA sequence encoded a cytosolic hexakinase in combination with DNA sequences ensuring the transcription and translation in plant cells.

31. A recombinant DNA molecule comprising a DNA sequence encoding a protein with the enzymatic activity of a hexokinase in combination with DNA sequence ensuring the transcription and translation in plant cells, wherein the S 20 hexokinase is a glucokinase.

32. Use of DNA sequences encoding an invertase in combination with DNA sequences encoding a hexokinase for the production of transgenic plant cells which, in S 25 comparison to non-transformed plant cells, exhibit an increased glycolysis rate.

33. The use of claim 33, wherein the hexokinase is a glucokinase.

34. A transgenic plant cell according to claim 1, substantially as hereinbefore described with reference to the examples.

35. A transgenic plant according to claim 19 or substantially as hereinbefore described with reference to Sthe examples. 35

36. A method according to claim 26, for the production of transgenic plant cells, substantially as hereinbefore described with reference to the examples.

37. A recombinant DNA molecule according to claim 31 or 32, substantially as hereinbefore described with reference to the examples. Dated this 7 th day of March 2000 PLANTTEC BIOTECHNOLOGIE GmbH By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia S S S o**