WO2004058962A1 - Method for producing plants containing starches with an increased phosphate content - Google Patents

Abstract

The present invention relates to a method for increasing the phosphate content of starches from genetically modified plant cells in comparison with corresponding unmodified wild-type plant cells, wherein a plant cell is genetically modified, with the genetic modification leading to a reduction of the activity of at least one phosphoglucomutase in comparison with corresponding wild-type plant cells which have not been genetically modified. There is furthermore described the use of one or more nucleic acid molecules, whose presence and/or expression leads to a reduction of the activity of at least one phosphoglucomutase in comparison with corresponding wild-type plant cells which have not been genetically modified, in order to increase the phosphate content in plant starch.

Description

METHOD FOR PRODUCING PLANTS CONTAINING STARCHES WITH AN INCREASED PHOSPHATE

CONTENT

Description

The present invention relates to a method for increasing the phosphate content in plant starches from genetically modified plants. The genetic modification consists in particular in the introduction of one or more foreign nucleic acid molecules whose presence, or expression, leads to a reduction of the activity of at least one phosphoglucomutase (PGM) in comparison with corresponding wild-type plant cells which has (have) not been genetically modified.

The polysaccharide starch is a polymer of chemically uniform units, the glucose molecules. However, it constitutes a highly complex mixture of different forms of molecules which differ with regard to their degree of polymerization and the occurrence of branches of the glucose chains. Starch therefore does not constitute a uniform raw material. Starch consists of two chemically different components, amylose and amylopectin. In typical plants used for starch production, such as, for example, maize, wheat or potato, amylose starch amounts to approximately 20% - 30% in the starch which has been synthesized and amylopectin starch to approximately 70% - 80%.

The functional properties of starch are affected greatly not only by the amylose/amylopectin ratio and the phosphate content, but also by the molecular weight, the pattern of the side-chain distribution, the ionic content, the lipid and protein content, the mean size of the starch grains, the starch grain morphology and the like. Important functional properties which may be mentioned in this context are, for example, solubility, the retrogradation behaviour, the water-binding capacity, the film-forming properties, the viscosity, the gelatinization properties, the freeze-thaw stability, the stability to acids, the gel strength and the like. The size of the starch grain too may be of importance for various applications.

An overview over various plant species which show a reduction in the enzymes which take part in starch biosynthesis is found in Kossmann and Lloyd (2000, Critical Reviews in Plant Sciences 19(3), 171-126).

Imported sucrose is degraded in the cytosol by sucrose synthase to give UDP-glucose and fructose. The resulting UDP-glucose is subsequently converted into glucose-1 -phosphate by UDP-glucose phyrophosphorylase (UGPase). The second product of the sucrose synthase reaction, namely fructose, is phosphorylated by fructokinase to give fructose-6-phosphate. Fructose-6-phosphate is converted into glucose-6-phosphate by phosphoglucose isomerase and subsequently into glucose-1 -phosphate by phosphoglucomutase. However, glucose-6-phosphate is the main precursor which is taken up by the amyloplast for starch synthesis (Tauberger et. al. 2000, Plant Journal 32(1), 43-53). Glucose-6-phosphate, in turn, is converted in the amyloplast by the plastid phosphoglucomutase to give glucose-1 -phosphate, which is converted by ADP-glucose pyrophosphorylase (AGPase; EC 2.7.7.27) to give ADP-glucose (Mϋller- Rober et al. 1992). This nucleotide sugar is the direct precursor for the starch synthases (SS; EC 2.4.1.21), which form -1 ,4-glycosidic bonds between the glucose units to give amylose. The branching enzymes (EC 2.4.1.24) are responsible for producing amylopectin; following hydrolytic cleavage of the α~1 ,4-glycosidic glucose chains, they generate α-1 ,6- giycosidic branching (Tauberger 1999, PhD thesis).

Plants in which the activity of a phosphoglucomutase is reduced have already been described. Work has focused on, for example, an increased sucrose content (WO 98-01574; EP1001029), a modified protein content (WO 01-75128) or the elucidation of the metabolic pathways (Tauberger 1999, PhD thesis; Tauberger et al 2000, Plant Journal 23(1), 43-53).

In comparison with corresponding wild-type plants, plants with a reduced phosphoglucomutase activity reveal an increased glucose-6-phosphate content, but also fructose-6-phosphate and glucose-1 -phosphate content (Tauberger 1999, loc. cit.). A trend to a reduced starch content and an increased sucrose and glucose accumulation is also observed.

WO 98-01574 describes pea plants in which the activity of the plastid phosphoglucomutase has been reduced owing to the mutation of an Rug3 enzyme and which have an increased sucrose content and a longer harvest window.

WO 01-75128 too relates to the production of legumes with a reduction of the activity of the plastid phosphoglucomutase. The aim is an increased protein content combined with a prolonged grain-filling period.

EP 1174510 describes plastid PGM sequences from maize, rice, soybeans and bullrush and the methodology for generating transgenic maize and soybeans with a modified plastid phosoglucomutase content.

A method for increasing the phosphate content in a plant starch by reducing the activity of at least one phosphoglucomutase in the plant (cell) used for starch production has not been described as yet.

The phosphate content can be modified in principle both by recombinant approaches and by the subsequent chemical phosphorylation of native starches (see, for example in: Starch Chemistry and Technology. Eds. R. L. Whistler, J. N. BeMiller and E. F. Paschall. Academic Press, New York, 1988, 349-364). However, chemical modifications are, as a rule, expensive and time-consuming and lead to starches whose physico- chemical properties may differ from in-vivo modified starches.

The starches produced by the method according to the invention furthermore differ from chemically phosphorylated starches by a modified phosphorylation pattern and, after gelatinization of the starches and gelling, by modified gel strength characteristics.

Novel functionalities are in great demand in the starch industry. Physical and/or chemical modifications of starch are required for a variety of applications. Starch suspensions with an increased phosphate content offer improved clarity and an improved low-temperature stability. Starches with an increased phosphate content permit the development of advantageous products mainly in papermaking, ready meals, dressings, dietetic products, in the baking industry and the like.

This object is achieved by providing the use forms specified in the patent claims.

Thus, it has been found, surprisingly, that a reduction of the activity of at least one phosphoglucomutase in a plant cell leads to an increased phosphate content of the starch produced.

In a first aspect, the present invention thus relates to a method for increasing the phosphate content in starches from genetically modified plant cells in comparison with corresponding unmodified wild-type plant cells, wherein a plant cell is genetically modified, with the genetic modification leading to a reduction of the activity of at least one phosphoglucomutase in comparison with corresponding wild-type plant cells which have not been genetically modified. A plant may be regenerated from, or using, the cells thus generated, using methods with which the skilled worker is familiar, and this plant can be used, if appropriate, for generating further plants.

The genetic modification can take the form of any genetic modification which leads to a reduction of the activity of one or more phosphoglucomutases which are endogenous to the plant cell in comparison with corresponding plant cells, of wild-type plants, which have not been genetically modified.

For the purposes of the present invention, the term "genetically modified" means that the genetic information of the plant cell is modified, the activity of at least one phosphoglucomutase being reduced. For example, genetically modified plant cells according to the invention show a reduction of the expression of one or more phosphoglucomutase genes which are endogenous to the plant cell in comparison with corresponding plant cells, of wild-type plants, which have not been genetically modified. Moreover, "genetically modified" is also understood as meaning a plant in which a reduction of the activity of at least one phosphoglucomutase has been brought about by means of a mutation. The mutants may take the form of either spontaneously occurring mutants or of mutants which have been generated by the targeted application of mutagens. Possibilities regarding mutagenesis will be described in greater detail hereinbelow. In the context of the present invention, "wild-type plants" or "wild-type plant cells" refers to those plants, or plant cells, which act as starting material in the method according to the invention, i.e. before the reduction according to the invention of the activity or expression of at least one phosphoglucomutase. As a rule, they take the form of plants or plant cells which are not genetically modified with regard to the activity or expression of phosphoglucomutase. However, genetically modified plants or plant cells which are genetically modified otherwise than regarding the activity or expression of phosphoglucomutase may also constitute the starting material according to the invention.

For the purposes of the present invention, the term "reduction of the activity" refers to a reduction of the enzymatic activity of a phosphoglucomutase protein in the cells and/or a reduction of the expression of endogenous genes encoding proteins of a phosphoglucomutase, and/or a reduction of the amount of phosphoglucomutase protein in the cells. For example, the genetic modification which has been carried out may also lead to the expression of inactive PGM protein in the plant cell. Details on the methodology of the activity determination are found under "General Methods".

In the context of the present invention, phosphoglucomutases are understood as meaning enzymes (E.C. 5.4.2.2.) which catalyze the conversion between glucose-1 -phosphate and glucose-6-phosphate (ap Rees and Morrell 1990, Am. Potato J. 67: 835-847). Proteins which have been identified as plastid phosphoglucomutases have already been isolated from spinach (Mϋhlbach and Schnarrenberger, 1978, Planta 141 : 65-70) and from pea (Salvucci et al., 1990, Plant Physiol. 93: 105-109). At least two PGM isoforms, a plastid form and a cytosolic form, exist in potato. The potato plastid PGM has a molecular mass of 62 kDa (Takamiya and Fukui 1978, Plant Cell Physiol. 19: 759-768). Tauberger (1999, loc. cit.) detected a constitutive expression of the plastid phosphoglucomutase (=aPGM II) in the main tissues of the potato plant such as leaves, shoot, tubers and roots by means of expression analysis. The cytosolic PGM, also referred to as PGM I, is indispensible for the sugar and starch metabolism and catalyzes the conversion reaction between glucose-1 -phosphate and glucose-6-phosphate. The potato cytosolic phosphoglucomutase (PGM I) is a protein of approx. 64 kDa and expressed constitutively in all main tissues of the potato plant. While the plastid isoform is important for providing glucose-1 -phosphate for starch biosynthesis in the amyloplasts, the cytosolic PGM ensures the supply of glucose-6-phosphate from glucose-1 -phosphate in the cytosol, firstly for transport into the amyloplasts and secondly also for glycolysis (energy supply). When the cytosolic PGM is inhibited, the concentrations of the soluble sugars sucrose and glucose remain unchanged.

Manjunath et al., 1998, (Plant Physiol. 117: 997-1006) isolated a cytosolic PGM from maize and studied its function.

In an advantageous embodiment according to the invention, the genetic modification can be brought about by introducing one or more foreign nucleic acid molecules into a plant cell.

In a further aspect, the present invention thus relates to a method for increasing the phosphate content in starches of genetically modified plants by introducing one or more foreign nucleic acid molecules into a plant cell. The presence and/or expression of these foreign nucleic acid molecules leads to the reduction of the activity of at least one phosphoglucomutase in comparison with corresponding unmodified plant cells from wild-type plants.

A plant can be regenerated from or using the genetically modified cells thus prepared, using customary methods, and, if appropriate, further plants can be generated from the above plant.

The reduction of the expression can be determined for example by measuring the amount of phosphoglucomutase-protein-encoding transcripts, for example by Northern Blot analsysis (Tauberger et al., 2000, The Plant Journal, 23(1), 43-53), quantitative RT-PCR or what are known as microarrays. "Reduced" in this context means a reduced amount of transcripts in comparison with corresponding cells which have not been genetically modified or wild-type cells, preferably by at least 50%, in particular by at least 70%, preference by at least 85% and especially preferably by at least 95%.

The reduction of the amount of phosphoglucomutase proteins, which is the result of the reduction of the activity of these proteins in the plant cells in question, can be determined for example by immunological methods such as Western Blot analysis, ELISA (Enzyme Linked Immuno Sorbent Assay) or RIA (Radio Immune Assay). These methods are known to the skilled worker and described in, for example, Sambrook et al., 1989, Molecular Cloning. A Laboratory Manual; Cold Spring Harbour Laboratory Press. "Reduced" means, in this context, a reduced amount of phosphoglucomutase protein in comparison with corresponding cells which have not been genetically modified or wild-type cells, preferably by at least 50%, in particular by at least 70%, by preference by at least 85% and especially preferably by at least 95%.

The determination of the phosphoglucomutase activity in total protein extracts is described in the chapter General Methods.

The terms "foreign nucleic acid molecule" or "foreign nucleic acid molecules" are understood as meaning, in the context of the present invention, such a molecule which either does not occur naturally in corresponding plant cells or which does not occur naturally in the plant cells in the specific spatial arrangement or which is localized at the location in the genome of the plant cell where it does not occur naturally. The foreign nucleic acid molecule is preferably a recombinant molecule which consists of various elements whose combination or specific spatial arrangement does not occur naturally in plant cells. Furthermore, the activity of at least one plastid phosphoglucomutase may be reduced by the method according to the invention in a preferred embodiment.

A plastid phosphoglucomutase is understood as meaning, in this context, an enzyme which catalyzes the conversion reaction between glucose-1 - phosphate and glucose-6-phosphate in the plastid. The plastid PGM plays an important role in the transport of glucose-6-phosphate into the amyloplasts and the supply of glucose-1 -phosphate. Moreover, it is another functionally important enzyme for the regulation of starch biosynthesis in storage tissue (Flϋgge and Weber, 1994, Planta 194: 181- 185; Flϋgge 1985, J. Experim. Botany 46: 1317-1323; Schott et al., 1995, Planta 196: 647-652; Kammerer et al, 1998, Plant Cell 10: 105-117).

However, a reduction of the activity of at least one cytosolic PGM may likewise take place within the scope of the present invention. In some cases it may be advantageous simuntaneously to reduce the activity of several PGM isoforms, for example cytosolic and plastid PGM. The genetic modifications required may be carried out simultaneously or in succession.

In a preferred embodiment, the increase in the phosphate content of the starch, which is brought about by the method according to the invention leads in particular to an increased phosphate content in the C-6 position of the starch produced thus in comparison with starch from corresponding wild-type plant cells which have not been genetically modified.

For the purposes of the present invention, the term "phosphate content" of the starch refers to the content of phosphate bonded covalently in the form of starch phosphate monoesters. In connection with the present invention, the term "increased phosphate content" means that the total phosphate content of covalently bonded phosphate and/or the phosphate content in the C-6 position of the starch synthesized in the plant cells according to the invention is increased in comparison with starch from plant cells of corresponding wild-type plants, preferably by at least 40%, by preference by at least 60%, especially preferably by at least 80%.

For the purposes of the present invention, the term "phosphate content in the C6 position" is understood as meaning the content of phosphate groups which are bound at the carbon atom position "6" of the glucose monomers of the starch. In principle, the positions C2, C3 and C6 of the glucose units in the starch may be phosphorylated in vivo. In connection with the present invention, the determination of the phosphate content in the C6 position (= C6-P content) can be performed via a glucose-6- phosphate determination using a visual-enzymatic assay (Nielsen et al., Plant Physiol. 105, (1994), 111-117).

The determination of the total phosphate content and the determination of the phosphate content in the C-6 position is described hereinbelow under "General Methods".

According to a further aspect according to the invention, the foreign nucleic acid molecule(s) introduced into the plant cell is, or are, selected from the group consisting of a) DNA molecules which encode at least one antisense RNA which brings about a reduction of the expression of at least one endogenous gene which encodes PGM protein(s); b) DNA molecules which, via a cosuppression effect, lead to a reduction of the expression of at least one endogenous gene which encodes PGM protein(s); c) DNA molecules which encode at least one ribozyme which specifically cleaves transcripts of at least one endogenous gene which encodes PGM protein(s); d) nucleic acid molecules introduced by means of in-vivo mutagenesis, which lead to a mutation or an insertion of a heterologous sequence in at least one endogenous gene encoding PGM protein(s), the mutation or insertion bringing about a reduction of the expression of said at least one gene or the synthesis of inactive PGM proteins; e) DNA molecules which simultaneously encode at least one antisense RNA and at least one sense RNA, where said antisense RNA and said sense RNA form a double-stranded RNA molecule which brings about a reduction of the expression of at least one endogenous gene encoding PGM protein(s) ; f) DNA molecules comprising transposons, the integration of the transposon sequences leading to a mutation or an insertion in at least one endogenous gene encoding PGM protein(s), which brings about a reduction of the expression of said at least one gene or the synthesis of inactive PGM proteins; and/or g) T-DNA molecules which, owing to insertion in endogenous genes encoding PGM protein, brings about a reduction of the expression of at least one gene encoding PGM protein(s) or the synthesis of inactive PGM proteins.

In this respect, the references mentioned in the present description in the explanation of the individual possibilities of reducing the PGM activity or expression are expressly incorporated herein by reference.

An example of a tool which may be used for inhibiting gene expression by means of antisense or cosuppression technology is a DNA molecule which encompasses all of the sequence encoding a phosphoglucomutase protein, including any flanking sequences, or else DNA molecules which only encompass parts of the coding sequence which, however, must be long enough in order to bring about an antisense effect, or cosuppression effect, in the cells. Sequences which are generally suitable for effective antisense or cosuppression inhibition have a minimum length of 15 bp, preferably a length of 100-500 bp; in particular sequences with a length of over 500 bp.

Another tool which is suitable for antisense or cosuppression approaches is the use of DNA sequences which have a high degree of homology with the sequences which occur endogenously in the plant cell and which encode phosphoglucomutase proteins. The minimum degree of homology should exceed approx. 65%. The use of sequences with at least 90%, in particular between 95% and 100% homology is to be preferred.

Another tool which is feasible for achieving an antisense or cosuppression effect is the use of introns, i.e. of noncoding regions of genes which encode phosphoglucomutase proteins.

The use of intron sequences for inhibiting the gene expression of genes which encode starch biosynthesis proteins has been described in international patent applications WO97/04112, WO97/04113, WO98/37213 und WO98/37214.

The skilled worker is familiar with the methods of achieving an antisense and cosuppression effect. For example, the cosuppression inhibition method has been described in Jorgensen (Trends Biotechnol. 8 (1990), 340-344), Niebel et al., (Curr. Top. Microbiol. Immunol. 197 (1995), 91- 103), Flavell et al. (Curr. Top. Microbiol. Immunol. 197 (1995), 43-46), Palaqui and Vaucheret (Plant. Mol. Biol. 29 (1995), 149-159), Vaucheret et al., (Mol. Gen. Genet. 248 (1995), 311-317), de Borne et al. (Mol. Gen. Genet. 243 (1994), 613-621). The expression of ribozymes to reduce the activity of certain enzymes in cells is likewise known to the skilled worker and described, for example, in EP-B-0321201. The expression of ribozymes in plant cells has been described, for example, in Feyter et al. (Mol. Gen. Genet. 250, (1996), 329-338).

Furthermore, the reduction of the phosphoglucomutase activity in the plant cells may also be achieved by what is known as "in-vivo mutagenesis", where an RNA-DNA hybrid oligonucleotide ("chimeroplast") is introduced into cells by transforming cells (Kipp, P.B. et al., Poster Session at the " 5^th International Congress of Plant Molecular Biology, 21st-27th September 1997, Singapore; R. A. Dixon and C.J. Arntzen, Meeting report to "Metabolic Engineering in Transgenic Plants", Keystone Symposia, Copper Mountain, CO, USA, TIBTECH 15, (1997), 441-447; international patent application WO 9515972; Kren et al., Hepatology 25, (1997), 1462- 1468; Cole-Strauss et al., Science 273, (1996), 1386-1389; Beetham et al., 1999, PNAS 96, 8774-8778).

A part of the DNA component of the RNA-DNA oligonucleotide is homologous to a nucleic acid sequence of an endogenous phosphoglucomutase gene, but, in comparison with the nucleic acid sequence of an endogenous phosphoglucomutase gene, contains a mutation or a heterologous region which is surrounded by the homologous regions.

The mutation or heterologous region which is present in the DNA component of the RNA-DNA oligonucleotide can be transferred into the genome of a plant cell by base pairing of the homologous regions of the RNA-DNA oligonucleotide and of the endogenous nucleic acid molecule, followed by homologous recombination. This leads to a reduction of the activity of one or more phosphoglucomutase proteins.

In the context of the invention, "genetic modification" may be understood as meaning the generation of plant cells according to the invention by subjecting one or more genes to mutagenesis. In this context, the nature of the mutation is irrelevant as long as it leads to a reduction of the activity of a phosphoglucomutase protein. In the context of the present invention, the term "mutagenesis" is to be understood as meaning any type of mutation such as, for example, deletion, point mutation (nucleotide substitution), insertion, inversion, gene conversion or chromosome translocation.

In this context, the mutation may be generated by the use of chemical agents or high-energy radiation (for example x-rays, neutron, gamma or UV radiation).

Agents which may be employed for generating chemically induced mutations, and the mutations resulting in this context by the action of the mutagens in question, have been described, for example, by Ehrenberg and Husain 1981 , (Mutation Research 86, 1-113) and Mϋller 1972 (Biologisches Zentralblatt 91 (1), 31-48). The generation of rice mutants using gamma-rays, ethylmethanesulfonate (EMS), N-methyl-N-nitrosurea or sodium azide (NaN₃) is described, for example, in Jauhar and Siddiq 1999, Indian Journal of Genetics, 59 (1), 23-28); in Rao 1977 (Cytologica 42, 443-450); Gupta and Sharma 1990 (Oryza 27, 217-219) and Satoh and Omura 1981 (Japanese Journal of Breeding 31 (3), 316-326). The generation of wheat mutants using NaN₃ or maleic hydrazide is described in Arora et al. (1992, Anals of Biology 8 (1), 65-69). A review over the generation of wheat mutants using various types of high-energy radiation and chemical agents can be found in Scarascia-Mugnozza et al. (1993, Mutation Breeding Review 10, 1-28). Svec et al. (1998, Cereal Research Communications 26 (4), 391-396) describes the use of N-ethyl-N- nitrosurea for generating mutants in triticale. The use of MMS and gamma radiation for generating millet mutants is described in Shashidhara et al. (1990, Journal of Maharashtra Agricultural Universities 15 (1), 20-23).

The generation of mutants in plant species which reproduce predominantly vegetatively has been described for example for potatoes which produce a modified starch (Hovenkamp-Hermelink et al. (1987, Theoretical and Applied Genetics 75, 217-221) and for mint with an increased oil yield or modified oil quality (Dwivedi et al., 2000, Journal of Medicinal and Aromatic Plant Sciences 22, 460-463). All of these methods are suitable in principle for generating the plant cells according to the invention and the starch produced by them.

The identification of mutations in the genes in question, in particular in genes encoding a phosphoglucomutase protein, may be effected with the aid of methods known to the skilled worker. Methods which may be employed in particular in this context are analyses based on hybridizations with probes (Southern blot), the amplification by means of polymerase chain reaction (PCI), the sequencing of relevant genomic sequences and the search for individual nucleotide substitutions. An example of a method for identifying mutations with the aid of hybridization patterns is the search for restriction fragment length polymorphisms (RFLP) (Nam et al., 1989, The Plant Cell 1 , 699-705; Leister and Dean, 1993, The Plant Journal 4 (4), 745-750). An example of a PCR-based method is the analysis of amplified fragment length polymorphisms (AFLP) (Castiglioni et al., 1998, Genetics 149, 2039-2056; Meksem et al., 2001 , Molecular Genetics and Genomics 265, 207-214; Meyer et al., 1998, Molecular and General Genetics 259, 150-160). The use of amplified fragments which have been cleaved with restriction endonucleases (cleaved amplified polymorphic sequences, CAPS) may also be used for identifying mutations (Konieczny and Ausubel, 1993, The Plant Journal 4, 403-410; Jarvis et al., 1994, Plant Molecular Biology 24, 685-687; Bachem et al., 1996, The Plant Journal 9 (5), 745-753). Methods for determining SNPs are, inter alia, by Qi et al. (2001 , Nucleic Acids Research 29 (22), 116) Drenkard et al. (2000, Plant Physiology 124, 1483-1492) and Cho et al. (1999, Nature Genetics 23, 203-207). Methods which are particularly suitable are those which permit a large number of plants to be studied for mutations in specific genes within short periods. Such a method, which is known as TILLING (targeting induced local lesions in genomes) has been described by McCallum et al. (2000, Plant Physiology 123, 439-442).

The use of all of these methods is suitable for the purposes of the present invention in order to generate plant cells and plants and the starch produced by them.

Moreover, the plant cells according to the invention may also be generated with the aid of homologous transposons, i.e. transposons which are naturally present in the plant cells in question. A review of the utilization of endogenous and heterologous transposons as tools in plant biotechnology can be found in Ramachandran and Sundaresan (2001 , Plant Physiology and Biochemistry 39: 234-252). The possibility of generating mutants with the aid of retrotransposons and methods for identifying mutants have been described by Kumar and Hirochika (2001 , Trends in Plant Science 6 (3), 127-134). The activity of heterologous transposons in different species has been described both for dicotyledonous plants and for monocotyledonous plants: for example for rice (Greco et al., 2001 , Plant Physiology 125, 1175-1177; Liu et al., 1999, Molecular and General Genetics 262, 413- 420; Hiroyuki et al., 1999, The Plant Journal 19 (5), 605-613; Jeon and Gynheung, 2001 , Plant Science 161 , 211-219), barley (2000, Koprek et al., The Plant Journal 24 (2), 253-263) Arabidopsis thaliana (Aarts et al., 1993, Nature 363, 715-717, Schmidt and Willmitzer, 1989, Molecular and General Genetics 220, 17-24; Altmann et al., 1992, Theoretical and Applied Genetics 84, 371-383; Tissier et al., 1999, The Plant Cell 11 , 1841-1852), tomato (Belzile and Yoder, 1992, The Plant Journal 2 (2), 173-179) and potato (Frey et al., 1989, Molecular and General Genetics 217, 172-177; Knapp et al., 1988, Molecular and General Genetics 213, 285-290).

In principle, the plant cells and plants according to the invention and the starch produced by them may be generated or produced with the aid of both homologous and heterologous transposons, the use of homologous transposons also being understood as meaning those which already naturally exist in the plant genome.

The T-DNA insertion mutagenesis is based on the ability of certain segments (T-DNA) of Ti plasmids from Agrobacterium to integrate into the genome of plant cells. The site of integration into the plant chromosome is not fixed, but may take place at any given site. If the T-DNA integrates into a segment of the chromosome which constitutes a gene function, the result may be a modified gene expression and thus modified activity of a protein encoded by the gene in question. In particular, the integration of a T-DNA into the coding region of a protein frequently leads to a situation where the protein in question can no longer be synthesized by the cell in question in active form, or not at all. The use of T-DNA insertions for generating mutants has been described for example for Arabidopsis thaiiana (Krysan et al., 1999, The Plant Cell 11 , 2283-2290; Atipiroz- Leehan and Feldmann, 1997, Trends in Genetics 13 (4), 152-156; Parinov and Sundaresan, 2000, Current Opinion in Biotechnology 11 , 157-161) and rice (Jeon and An, 2001 , Plant Science 161 , 211-219; Jeon et al., 2000, The Plant Journal 22 (6), 561-570). Methods for identifying mutants which have been generated with the aid of T-DNA insertion mutagenesis have been described, inter alia, by Young et al., (2001 , Plant Physiology 125, 513-518), Parinov et al. (1999, The Plant Cell 11 , 2263-2270), Thorneycroft et al. (2001 , Journal of Experimental Botany 52, 1593-1601), and McKinney et al. (1995, The Plant Journal 8 (4), 613-622.

Moreover, the reduction of the phosphoglucomutase activity in plant cells may also be brought about by the simultaneous expression by sense and antisense molecules of the target gene to be repressed in each case, preferably the phosphoglucomutase gene.

This may be achieved for example by using chimeric constructs which contain "inverted repeats" of the target gene, or parts of the target gene, in question. The chimeric constructs encode sense and antisense RNA molecules of the target gene in question. Sense and antisense RNA are synthesized simultaneously in planta as one RNA molecule, it being possible for sense and antisense RNA to be separated from each other by means of a spacer and to form a double-stranded RNA molecule.

It has been possible to demonstrate that introduction of inverted-repeat DNA constructs into the genome of plants is a highly effective method for repressing the genes which correspond to the inverted-repeat DNA constructs (Waterhouse et al., Proc. Natl. Acad. Sci. USA 95, (1998),

13959-13964; Wang and Waterhouse, Plant Mol. Biol. 43, (2000), 67-82;

Singh et al., Biochemical Society Transactions Vol. 28 part 6 (2000), 925- 927; Liu et al., Biochemical Society Transactions Vol. 28 part 6 (2000),

927-929); Smith et al., (Nature 407, (2000), 319-320; international patent application WO99/53050 A1). Sense and antisense sequences of the target gene, or target genes, may also be expressed separately from one another by means of the same or different promoters (Nap, J-P et al, 6^th International Congress of Plant Molecular Biology, Quebec, 18th-24th

June, 2000; Poster S7-27, Session S7). It is furthermore known that the formation of double-stranded RNA molecules of promoter DNA molecules in plants in trans may lead to methylation, and transc ptional inactivation, of homologous copies of these promoters, which shall hereinbelow be referred to as target promoters (Mette et al., EMBO J. 19, (2000), 5194-5201).

Thus, it is possible, via inactivating the target promoter, to reduce the gene expression of a specific target gene (for example phosphoglucomutase gene) which is naturally under the control of this target promoter, i.e. the DNA molecules which comprise the target promoters of the genes to be repressed (target genes) are in this case - in contrast to the original function of promoters in plants - used as DNA molecules which can be transcribed themselves and not as control elements for the expression of genes or cDNAs.

Constructs which are preferably used for generating the double-stranded target promoter RNA molecules in planta, where they may be present as RNA hairpin molecules, are those which contain "inverted repeats" of the target promoter DNA molecules, the target promoter DNA molecules being under the control of a promoter which governs the gene expression of said target promoter DNA molecules. These constructs are subsequently introduced into the genome of plants. The expression of the "inverted repeats" of said target promoter DNA molecules results in the formation of double-stranded target promoter RNA molecules in planta (Mette et al., EMBO J. 19, (2000), 5194-5201). The target promoter may thus be inactivated.

The skilled worker furthermore knows that the activity of PGM proteins can be achieved by expressing nonfunctional derivatives, in particular trans- dominant mutants, of such proteins and/or by expressing antagonists/inhibitors of such proteins.

Antagonists/inhibitors of such proteins encompass for example antibodies, antibody fragments or molecules with similar binding properties. For example, a cytoplasmic scFv antibody was employed for modulating the activity of the phytochrome A protein in genetically modified tobacco plants (Owen, Bio/Technology 10 (1992), 790-4; Review: Franken, E, Teuschel, U. and Hain, R., Current Opinion in Biotechnology 8, (1997), 411-416; Whitelam, Trends Plant Sci. 1 (1996), 268-272).

Meaningful promoters for the expression of nucleic acids which reduce the activity of a target gene are, for example, the cauliflower mosaic virus 35S RNA promoter and the maize ubiquitine promoter for constitutive expression, the Patatin gene promoter B33 (Rocha-Sosa et al., EMBO J. 8 (1989), 23-29), the MCPI promoter of the potato metallocarbopeptidase inhibitor gene (Hungarian patent application HU9801674) or the potato GBSSI promoter (international patent application WO 92/11376) for a tuber-specific expression in potatoes, or a promoter which ensures expression only in photosynthetically active tissues, for example the ST- LS1 promoter (Stockhaus et al., Proc. Natl. Acad. Sci. USA 84 (1987), 7943-7947; Stockhaus et al., EMBO J. 8 (1989), 2445-2451), the Ca/b promoter (see, for example, US 5656496, US 5639952, Bansal et al., Proc. Natl. Acad. Sci. USA 89, (1992), 3654-3658) and the Rubisco SSU promoter (see, for example, US 5034322, US 4962028), or, for endosperm-specific expression, the glutelin promoter (Leisy et al., Plant Mol. Biol. 14, (1990), 41-50; Zheng et al., Plant J. 4, (1993), 357-366; Yoshihara et al., FEBS Lett. 383, (1996), 213-218), the Shrunken-1 promoter (Werr et al., EMBO J. 4, (1985), 1373-1380), the wheat HMG promoter, the USP promoter, the phaseolin promoter, or promoters of maize zein genes (Pedersen et al., Cell 29, (1982), 1015-1026; Quatroccio et al., Plant Mol. Biol. 15 (1990), 81-93).

The expression of the foreign nucleic acid molecule(s) is particularly advantageous in those plant organs which store starch. Such organs are, for example, the tuber of the potato plant or the grains, or the endosperm, of maize, wheat or rice plants. It is therefore preferred to use promoters which mediate the expression in these organs.

However, it is also possible to use promoters which are only activated at a point in time which is determined by external influences (see, for example, WO 93/07279). Promoters of heat-shock proteins, which permit simple induction, may be of particular interest in this context. Furthermore, seed- specific promoters, such as, for example, the Vicia faba USP promoter, which ensures seed-specific expression in Vicia faba and other plants, may be (Fiedler et al., Plant Mol. Biol. 22, (1993), 669-679; Baumlein et al., Mol. Gen. Genet. 225, (1991), 459-467). Others which may be employed are fruit-specific promoters, such as, for example, described in WO91/01373. Furthermore, a termination sequence which serves for the correct termination of the transcription process and the addition of a poly-A tail to the transcript, which is assumed to have a function in stabilizing the transcripts, may be present. Such elements have been described in the literature (cf., for example, Gielen et al., EMBO J. (1989), 8:23-29) and can be exchanged as desired.

The genetically modified plant cells generated by the method according to the invention synthesize a modified starch whose physico-chemical properties, in particular the phosphate content, the viscosity behavior and/or the gel strength, is modified in comparison with starch synthesized in wild-type plants so that it is better suited to specific uses. Surprisingly, it has been found that the starch of the plant cells generated by the present method has an increased phosphate content in comparison with starch from plant cells of corresponding wild-type plants, so that this starch is better suited to specific uses.

By gel strength, the skilled worker means the resistance of a starch gel to deformation under specific conditions. Gel strength can be measured for example by measuring the force required when a standardized measuring rod is immersed into a starch gel, or the force required for pulling a standardized measuring rod out of a starch gel is estimated.

In connection with the present invention, the gel strength shall be determined with the aid of a Texture Analyzer under the conditions described in the methodological part.

In the context of the present invention, the amylose content is determined by the method of Hovenkamp-Hermelink et al. (Potato Research (1988), 31 :241-246) which has been described below for potato starch. This method can also be applied to isolated starches of other plant species. Methods for isolating starches are known to the skilled worker.

In a further aspect, the present invention relates to the use of one or more nucleic acid molecules for increasing the phosphate content in plant starch, wherein a plant cell is genetically modified, the genetic modification leading to a reduction of the activity of at least one phosphoglucomutase in comparison to corresponding wild-type plant cells (which have not been genetically modified).

In a preferred embodiment according to the invention, the at least one nucleic acid molecule used in this context is selected among the groups illustrated hereinabove. In a further preferred embodiment according to the invention, the use according to the invention of one or more nucleic acid molecules leads to an increased phosphate content in the C6 position of the starch in comparison with starch of corresponding wild-type plant cells (which have not been genetically modified). In a preferred embodiment, the at least one phosphoglucomutase whose expression and/or activity has been reduced may be a plastid phosphoglucomutase.

The starch obtained by the method according to the invention preferably exhibits an increased gel strength.

For the purposes of the present invention, the term "increased gel strength" refers to an increase in gel strength preferably by at least 30%, in particular by at least 50%, by preference by at least 70% and especially preferably by at least 100%, by a maximum of not more than 150% or not more than 200% in comparison with the gel strength of starch of corresponding wild-type plant cells (which have not been genetically modified).

In a further embodiment of the present invention, the modified starch produced by the method according to the invention is not only distinguished by an increased phosphate content in comparison with starch from corresponding wild-type plants, but also by a modified side- chain distribution.

In a further embodiment, the present invention relates to genetically modified plant cells which synthesize a modified starch, the modified starch being characterized in that it has an modified side-chain distribution

In one embodiment of the present invention, the term "modified side-chain distribution" is understood as meaning an increase of the amount of amylopectin side chains with a dp (= degree of polymerization) of 6 and 7 by in total at least 50%, preferably by at least 100%, especially by at least 125% and especially preferably by at least 150% in comparison with the amount of the corresponding amylopectin side chains from wild-type plants.

In a further embodiment according to the invention, the amount of amylopectin side chains with a dp of 8 to 15 in the starch produced by the method according to the invention is increased by at least 5%, by preference at least 10%, in particular by at least 20%. Furthermore, the amount of the side chains with a dp of 16-23 in the starch produced by the method according to the invention can preferably be reduced by at least 10%, by preference by at least 15%, in particular by at least 20% in comparison with the amount of the corresponding amylopectin side chains in wild-type plants.

The method according to the invention for the production of a starch with increased phosphate content encompasses the extraction of the starch from a plant and/or from starch-storing parts of such a plant and/or from a plant cell of such a plant and/or from propagation material of such a plant, said plant having been generated by the above-described method for increasing the phosphate content by reducing the activity of at least one phosphoglucomutase in genetically modified plant cells.

These plant cells may belong to any plant species, i.e. both to monocotyledonous and dicotyledonous plants. They are preferably plant cells from agriculturally useful plants, i.e. plants which are grown by man for the purposes of nutrition or for technical, particularly industrial, purposes. The invention preferably relates to fiber-developing plants (for example flax, hemp, cotton), oil-storing plants (for example oilseed rape, sunflower, soybean), sugar-storing plants (for example sugarbeet, sugarcane, sugar millet) and protein-storing plants (for example legumes).

In a further preferred embodiment, the invention relates to plant cells from starch-storing plants (for example, wheat, barley, oats, rye, potatoes, maize, rice, pea, cassava); potato plant cells are especially preferred.

A variety of techniques are available for introducing DNA into a plant host cell. These techniques encompass the transformation of plant cells with T- DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as transformation agent, protoplast fusion, injection, the electroporation of DNA, the introduction of DNA by means of the biolistic approach and other possibilities.

The use of agrobacteria-mediated transformation of plant cells has been studied intensively and described sufficiently in EP 120516; Hoekema, IN: The Binary Plant Vector System Offsetdrukkerij Kanters B.V., Alblasserdam (1985), Chapter V; Fraley et al., Crit. Rev. Plant Sci. 4, 1-46 and in An et al. EMBO J. 4, (1985), 277-287. As regards the transformation of potato, see, for example, Rocha-Sosa et al., EMBO J. 8, (1989), 29-33.).

The transformation of monocotyledonous plants by means of vectors based on agrobacterial transformation has also been described (Chan et al., Plant Mol. Biol. 22, (1993), 491-506; Hiei et al., Plant J. 6, (1994) 271- 282; Deng et al, Science in China 33, (1990), 28-34; Wilmink et al., Plant Cell Reports 11 , (1992), 76-80; May et al., Bio/Technology 13, (1995), 486-492; Conner and Domisse, Int. J. Plant Sci. 153 (1992), 550-555; Ritchie et al, Transgenic Res. 2, (1993), 252-265). An alternative system for the transformation of monocotyledonous plants is the transformation by means of the biolistic approach (Wan and Lemaux, Plant Physiol. 104, (1994), 37-48; Vasil et al., Bio/Technology 11 (1993), 1553-1558; Ritala et al., Plant Mol. Biol. 24, (1994), 317-325; Spencer et al., Theor. Appl. Genet. 79, (1990), 625-631), protoplast transformation, the electroporation of partially permeabilized cells, and the introduction of DNA by means of glass fibers. The transformation of maize, in particular, is described repeatedly in the literature (cf., for example WO95/06128, EP0513849, EP0465875, EP0292435; Fromm et al., Biotechnology 8, (1990), 833-844; Gordon-Kamm et al., Plant Cell 2, (1990), 603-618; Koziel et al., Biotechnology 11 (1993), 194-200; Moroc et al., Theor. Appl. Genet. 80, (1990), 721-726).

The successful transformation of other cereal species has also been described, for example for barley (Wan and Lemaux, loc. cit.; Ritala et al., loc. cit.; Krens et al., Nature 296, (1982), 72-74) and for wheat (Nehra et al., Plant J. 5, (1994), 285-297). All of the abovementioned methods are suitable for the purposes of the present invention.

In general, any promoter which is active in plant cells is suitable for expressing the foreign nucleic acid molecule(s). The promoter may be chosen in such a way that expression in the plants according to the invention takes place constitutively or only in a specific tissue, at a specific point in time of plant development or at a point in time determined by external influences. With regard to the plant, the promoter may be homologous or heterologous.

List of figures

Figure 1 shows a graphic representation of the change in the amylopectin side chain distribution in comparison with the wild type (WT). The starch analyzed was isolated from potato tubers of wild-type plants (250JP Desi) and plants with a reduced activity of a phosphoglucomutase protein (250JP005 (= aPGM2), 250JP026 (aPGM3) and 250JP060 (=aPGM4)). Fig. 2 shows a schematic representation of vector pA7 Fig. 3 shows a schematic representation of vector JP 6-101 Fig. 4 shows a schematic representation of vector pBinAr-Met Fig. 5 shows a schematic representation of vector JP 7-116

Abbreviations used in the figures:

35S: cauliflower mosaic virus promoter OCS: polyadenylation sequence (terminator), Agrobacterium tumefaciens octopine synthase gene

ORI: origin

AMP: ampicillin resistance

LB: left border RB: right border

Dhfr: dehydrofolate reductase (resistance to methodrexate)

Nos: omoter I Agrobacterium tumefaciens nopaline synthase.

Ag7: polyadenylation sequence

List of sequences:

Seq ID 1 : Nucleic acid sequence amplified from the published sequence of the plastid phosphoglucomutase (AC: AJ240053) with the primers JP-1a (5'-TTT gTC gAC ATC CAC ACg AgT TTC AAT TCC-3') and JP-1b (5'-ggg CAA CgC gAT CTA gAg AAC C-3') by RT-PCT technology as a 1.2 kb DNA pPGM2 fragment from total RNA from potato tubers;

Seq ID 2: Amino acid sequence of the plastid phosphoglucomutase from potato of Seq ID 1 above. General methods

The following methods were used in the examples:

Starch analysis a) Determination of the amylose content and of the amylose/amylopectin ratio

Starch was isolated from potato plants by standard methods, and the amylose/amylopectin ratio was determined by the method described by

Hovenkamp-Hermelink et al. (Potato Research 31 , (1988), 241-246).

b) Determination of the phosphate content

In starch, the positions C2, C3 and C6 of the glucose units may be phosphorylated. To determine the C6-P content of the starch, 50 mg of starch are hydrolyzed for 4 hours at 95°C in 500 μl of 0.7 M HCI. The mixtures are subsequently centrifuged for 10 minutes at 15500 g and the supematants removed. 7 μl of the supematants are mixed with 193 μl of imidazole buffer (100 mM imidazole, pH 7.4; 5 mM MgCI₂, 1 mM EDTA and 0.4 mM NAD). The measurement was carried out in a photometer at

340 nm. After a basic absorption had been established, the enzyme reaction was started by addition of 2u glucose-6-phosphate dehydrogenase (from Leuconostoc mesenteroides, Boehringer

Mannheim). The change in absorption is directly proportional to the concentration of the G-6-P content of the starch.

The total phosphate content was determined by the method of Ames (Methods in Enzymology VIII, (1966), 115-118).

Approximately 50 mg starch are treated with 30 μl of ethanolic magnesium nitrate solution and ashed for 3 hours at 500°C in the muffle furnace. The residue is treated with 300 μl of 0.5 M hydrochloric acid and incubated for 30 min at 60°C. An aliquot portion is subsequently made up to 300 μl with 0.5 M hydrochloric acid, and this is added to a mixture of 100 μl of 10% strength ascorbic acid and 600 μl of 0.42% strength ammonium molybdate in 2 M sulfuric acid and incubated for 20 min at 45°C. A photometric determination at 820 nm is now carried out, using a phosphate calibration series as the standard.

c) Determination of the gel strength (Texture Analyser) 1.5 g of starch (TS) are gelatinized in 25 ml of an aqueous suspension in the RVA apparatus (temperature program: see item d) "determination of the viscosity properties using a Rapid Visco Analyzer (RVA)") and subsequently kept in a sealed vessel for 24 hours at room temperature. The samples are fixed under the probe (cylindrical piston with planar surface) of a Texture Analyser TA-XT2 from Stable Micro

Systems (Surrey, UK), and the gel strength was determined using the following parameters:

Test speed 0.5 mm/s

Depth of penetration 7 mm - Contact surface 113 mm²

Pressure 2 g

d) Determination of the viscosity properties by means of a Rapid Visco Analyzer (RVA)

2 g of starch (TS) are taken up in 25 ml of H₂O (DM-type water, conductivity at least 15 mega Ohm) and used for analysis in a Rapid Visco Analyzer (Newport Scientific Pty Ltd., Investmet Support Group, Warriewod NSW 2102, Australia). The apparatus was operated in accordance with the manufacturer's instructions. The individual steps were carried out as follows: Step 1 : First, the starch suspension was stirred for 10 minutes at 960 rpm and subsequently heated, initially for one minute, at 50°C at a measuring rate of 160 rpm.

Step 2: The temperature is increased from 50°C to 95°C at a heating rate of 12°C per minute.

Step 3: The temperature is kept at 95°C for 2.5 minutes Step 4: The starch suspension is cooled from 95°C to 50^CC in steps of 12°C per minute

Step 5: The temperature is held at 50°C for 2 minutes. After the program has finished, the stirrer is removed and the beaker covered. The gelatinized starch is now available for texture analysis after 24 hours.

The profile of the RVA analysis contains characteristic values which are shown for comparing different measurements and substances. The following terms are to be understood as follows in connection with the present invention:

1. Maximum viscosity (RVA Max)

The maximum viscosity is understood as meaning the highest viscosity value measured in cP (centipoise) which is obtained in step 2 or 3 of the temperature profile.

2. Minimum viscosity (RVA Min)

The minimum viscosity is understood as meaning the lowest viscosity value, measured in cP, which is observed in the temperature profile after the maximum viscosity. This normally takes place in step 3 of the temperature profile.

3. Final viscosity (RVA Fin)

The final viscosity is understood as meaning the viscosity value, measured in cP, which is observed at the end of the measurement.

4. Setback (RVA Set) What is known as the "setback" is calculated by subtracting the value of the final viscosity from that of the setback. 5. Peak temperature (RVA T)

The peak temperature is understood as meaning the temperature in the temperature profile at which the viscosity first increases by 25 cP over a period of 20 sec.

e) Analysis of the amylopectin side chain distribution by means of ion- exchange chromatography

To separate amylose and amylopectin, 200 mg of starch are dissolved in 12 ml 90% (v/v) DMSO in H₂0 in 50 ml reaction vessels. After addition of 3 volumes of ethanol, the precipitate is separated by centrifugation for 10 minutes at approximately 1800 g at room temperature (RT). The pellet is then washed with 30 ml of ethanol, dried and dissolved in 40 ml of 1 % (w/v) NaCI solution at 75°C. After the solution has cooled to 30°C, approximately 90 mg of thymol are added slowly, and this solution is incubated for at least 60 hours at 30°C. The solution is subsequently centrifuged for 30 minutes at 2000 g (RT). The supernatant is then treated with 3 volumes of ethanol, and the amylopectin which precipitates is separated by centrifugation for 5 minutes at 2000 g (RT). The pellet (amylopectin) is then washed with ethanol and dried using acetone. A 1% strength solution is prepared by adding DMSO to the pellet, of which 200 μl are treated with 345 μl of water, 10 μl of 0.5 M sodium acetate (pH 3.5) and

5 μl of isoamylase (dilution 1 :10; Megazyme) and incubated for approximately 16 hours at 37°C. An aqueous 1 :5 dilution of this digest is subsequently filtered using a 0.2 μm filter, and 100 μl of the filtrate are analyzed by ion chromatography (HPAEC-PAD, Dionex). Separation was carried out using a PA-100 column (equipped with a suitable precolumn), while detection was carried out amperometrically.

The elution conditions were as follows:

Solution A - 0.15M NaOH

Solution B - 1 M sodium acetate in 0.15M NaOH

The relative amount of short side chains in the total of all side chains is determined via the determination of the percentage of a particular side chain in the total of all side chains. The total of all side chains is determined via the determination of the total area under the peaks which represent the degrees of polymerization 6 to 23 in the HPCL chromatogram.

The percentage of a specific side chain in the total of all side chains is determined via the determination of the ratio of the area under the peak which represents this side chain in the HPLC chromatogram to the total area. The program Chromelion 6.20 Version 6.20 from

Dionex, USA, was used for determining the peak areas.

rotein extraction for the determination of enzyme activities

The protein was extracted following a modified method of Geigenberger and Stitt 1993 (Planta 189: 329-339). Leaf material

(approx. 30-35 mg) or tuber material (approx. 80-120 mg) was homogenized in the frozen state (Heidolph, Kehlheim) and treated with 500 μl of extraction buffer (50 mM Hepes-KOH, pH 7.4; 5 mM MgCI₂; 1 mM EDTA; 1 mM EGTA; 5 mM DTT; 3 mM benzamidine; 2 mM ε- Amoni-n-caproic acid; 0.5 mM phenylmethylsulfonyl fluoride; 0.1 % (v/v) Triton X-100; 10% glycerol). The extracts were desalted using NAP™5 columns (Pharmacia Biotech,

Freiburg), divided into aliquot portions and frozen in liquid nitrogen. They were stored at -80°C.

g) Determination of the phosphoglucomutase activity in total protein extracts

The phosphoglucomutase activity was determined following the method of Galloway and Dugger (1994, Physiol. Plantarum 92: 479-486). The activity assay was carried out in a buffer consisting of 80 mM Hepes-

NaOH (pH 7.4), 10 μM glucose-1 , 6-bisphosphate, 10mM MG acetate, 15 mM L-cysteine, 0.6 mM NAD and 0.15 u glucose-6-phosphate dehydrogenase (Leuconostoc mesenteroides) with addition of 20 μl of total protein extract. The reaction was started by substrate addition of 10 mM glucose-1 -phosphate. The enzyme-coupled reaction, which can be measured spectrophotometrically, proceeded in each case in a final volume of 300 μl and was measured in the microtiter plate reader Spectramax (Molecular Devices Corp., Sunnyvale, Cal.) at a wavelength of 340 nm. The Vmax was determined using the associated software "Biolise". The phosphoglucomutase activity was computed using Microsoft Excel 97 and indicated in μmol/min/g fresh weight +/- standard error. Examples

Generation of transgenic potato plants with a reduced gene expression of a plastid phosphoglucomutase (aPGM II)

To generate transgenic plants with a reduced activity of an aPGM II protein, the T-DNA of the plasmid (JP7-116) was transferred into potato plants (var. Desiree) with the aid of agrobacteria as described by Rocha- Sosa et al. (EMBO J., (1989), 8:23-29).

Example 1

Preparation of the expression vector JP 7-116

To construct the vector JP 7-116, the primers JP-1a (5'-TTT gTC gAC ATC CAC ACg AgT TTC AAT TCC-3') and JP-1 b (5'-ggg CAA CgC gAT CTA gAg AAC C-3') were designed using the published sequence of the plastid phosphoglucomutase (AC: AJ240053), and a 1.2 kb DNA pPGM2 fragment from the total RNA from potato tubers was amplified by means of RT-PCT technique (Stratagene ProSTAR™ HF single-tube RT-PCR system). The resulting DNA fragment (see SEQ ID No. 1) was subsequently subcloned in antisense orientation based on the promoter into the Sall-Xbal cleavage site of the cloning vector pA7 in the form of an Sall-Xbal fragment (Fig. 2, compare description of the vector pBinAR). The resulting vector was termed JP 6-101 (Fig. 3).

The vector pBinAR-Met was used for the subsequent cloning. The vector pBinAR-Met originates from plasmid pGPTV-DHFR (Becker et al. 1992, Plant Mol. Biol. 20: 1195-1197), which constitutes a derivative of the vector pBin19. Instead of the nptll gene, pBinAR-Met contains the dhfr gene, which confers resistance to methotrexate, and instead of the 3' end of the nopalin synthase gene it contains the 3' end of gene 7 of the T-DNA of the Ti plasmid pTiACHδ (Nukleotide 2106-2316; Gielen et al., 1984, EMBO J 3: 835-846). Starting from plasmid pA7 (compare description of the vector pBinAR), the EcoRI-/-//77dlll fragment, comprising the 35S promoter, the ocs terminator and the interposed portion of the polylinker was ligated into the suitably cleaved plasmid pGPTV-DHFR. The resulting vector was referred to as pBinAR-Met (Fig. 4).

The plasmid pBinAR is a derivative of the vector plasmid pBin19 (Bevan 1984, Nucl Acids Res 12: 8711-8721 ; Frisch et al., 1995, Plant. Mol. Biol. 27:405-409; GenBank U09365) and was constructed as follows: a 529 bp fragment, which encompasses the nucleotides 6909-7437 of the cauliflower mosaic virus 35S RNA promoter, was isolated from the plasmid pDH51 (Pietrzak et al. 1986, Nucl. Acids Res. 14:5857-5868) as an EcoR\/Kpn\ fragment and ligated between the EcoRI and Kpn\ cleavage sites of the pUC18 polylinker. This gave rise to the plasmid pUC18-35S.

A 192 bp fragment which encompasses the polyadenylation signal (3' end) of the octopine synthase gene (gene 3) of the T-DNA of the Ti plasmid pTiACHδ (Gielen et al., 1984, loc. cit.) (nucleotides 11749-11939) was isolated from the plasmid pAGV40 (Herrera-Estrella et al. 1983, Nature 303:209-213) with the aid of the restriction endonucleases Hind\\\ and PvuW. Following the addition of Ssp\ linkers to the PvuW cleavage site, the fragment was ligated between the Sph\ and Hind\\\ cleavage sites of pUC18-35S. This gave rise to the plasmid pA7.

The entire polylinker comprising the 35S promoter and the ocs terminator was excised from pA7 using EcoRI and Hind\\\ and ligated into the suitably cleaved pBin19. This gave rise to the plant expression vector pBinAR (Hδfgen and Willmitzer 1990, Plant Science 66:221-230). In the vector pBinAR-Met, the 35S promoter OCS cassette was replaced by the EcoRI-Hindlll cassette from vector JP 6-101 , which cassette consisted of the 35S promoter, pPGM2 and OCS terminator, via the EcoRI-Hindlll cleavage sites of the vector. The resulting vector was termed JP 7-116 (Fig.5 ).

A variant of the method of Rocha-Sosa et al. (1989, EMBO J. 8: 23-29) was employed for the transformation of Solanum tuberosum L. cv. Desiree (Dietze et al., 1995, Gene transfer to plants. Potrykus, I. and Spangenberg, G (eds.), Springer-Verlag Berlin, 24-29).

The reduced activity of the endogenous PGM in the plants obtained by transformation with the plasmid JP 7-116, which were referred to as aPGM, was confirmed using the above-described methods. Tissue samples of tubers from the independent transformants were taken and the C-6 phosphate contents were determined (see Methods). The starches of the independent lines whose tubers showed the highest C-6 phosphate contents were used for a further analysis of the starch characteristics.

Process of extracting starch from potatoes

All of the tubers of one line (4 to 5 kg) are processed together in a commercially available juice extractor (Multipress automatic MP80, Braun). The starch-containing water of the fruits is collected in a 10-L bucket containing 200 ml of tap water with a spoon-tipful (approx. 3-4 g) of sodium disulfite. Thereafter, the bucket is filled completely with tap water. After allowing the starch to settle for 2 hours, the supernatant is decanted off and the starch is suspended again in 10 1 of tap water and poured through a sieve of mesh size 125 μm. After 2 hours (starch has again settled on the bottom of the bucket), the aqueous supernatant is again decanted off. This wash procedure is repeated 3 more times, so that the starch is resuspended five times in total in fresh tap water. Thereafter, the starches are dried at 37°C to a water content of 12-17% and homogenized in a mortar. The starches are now available for analyses.

Example 2

Analysis of the starch from plants with reduced aPGM II gene expression

The starch of various independent lines originating from the transformation described in Example 1 was isolated from potato tubers. The physico- chemical characteristics of this starch were subsequently analyzed. The results of the characterization of the modified starches are shown in Table 1 (Tab. 1) for a selection of certain plant lines which act as an example.

Percentage compared to wild type

Tab. 1 Summary of the physico-chemical characteristics:

Top: Absolute values obtained in the RVA analysis of starch isolated from wild-type plants (cv. Desiree) and plants with a reduced activity of a phosphoglucomutase (aPGM2 (250JP 005), aPGM3 (250JP 026) and aPGM4 (250JP 060)) relative to the values for the wild-type starch.

Bottom: Percentage values from the RVA analysis of starch isolated from wild-type plants (cv. Desiree) and plants with a reduced activity of a phosphoglucomutase (aPGM2 (250JP 005), aPGM3 (250JP 026) and aPGM4 (250JP 060)) relative to the values for the wild-type starch.

The analysis of the amylopectin side-chain distribution was carried out as described above. The table which follows (Tab. 2) contains an overview over the individual peak areas of the HPAEC chromatogram in relation to the total peak area of wild-type plants (cv. Desiree) and of aPGM II plants.

Tab. 2: Amylopectin side-chain distribution

The analysis of the amylopectin side-chain distribution was carried out as described above. The table contains an overview over the individual peak areas of the HPAEC chromatogram in relation to the total peak areas of wild-type plants (Desiree) and of aPGM2 (250JP 005), aPGM3 (250JP 026) and aPGM4 (250JP 060) plants (potato plants with a reduced activity of an aphosphoglucomutasell protein). The number of glucose monomers in the individual side chains is shown as dp.

Claims

Patent Claims

1. Method for increasing the phosphate content in starches from genetically modified plant cells in comparison with corresponding unmodified wild-type plant cells, wherein a plant cell is genetically modified, with the genetic modification leading to a reduction of the activity of at least one phosphoglucomutase in comparison with corresponding wild-type plant cells which have not been genetically modified.

2. Method according to Claim 1 , characterized in that the plant cell is genetically modified by the introduction of one or more foreign nucleic acid molecules whose presence and/or expression leads to the reduction of the activity of at least one phosphoglucomutase in comparison with corresponding wild-type plant cells which have not been genetically modified.

3. Method according to Claim 1 or 2, characterized in that the at least one phosphoglucomutase is a plastid phosphoglucomutase.

4. Method according to Claims 1 to 3, characterized in that the increase of the phosphate content of the starch leads to an increased content of phosphate in the C6 position of the starch.

5. Method according to one of Claims 1-4, characterized in that said foreign nucleic acid molecule is selected from the group consisting of: a) DNA molecules which encode at least one antisense RNA which brings about a reduction of the expression of at least one endogenous gene which encodes PGM protein(s); b) DNA molecules which, via a cosuppression effect, lead to a reduction of the expression of at least one endogenous gene which encodes PGM protein(s); c) DNA molecules which encode at least one ribozyme which specifically cleaves transcripts of at least one endogenous gene which encodes PGM protein(s); d) nucleic acid molecules introduced by means of in-vivo mutagenesis, which lead to a mutation or an insertion of a heterologous sequence in at least one endogenous gene encoding PGM protein(s), the mutation or insertion bringing about a reduction of the expression of said at least one gene or the synthesis of inactive PGM proteins; e) DNA molecules which simultaneously encode at least one antisense RNA and at least one sense RNA, where said antisense RNA and said sense RNA form a double-stranded RNA molecule which brings about a reduction of the expression of at least one endogenous gene encoding PGM protein(s) ; f) DNA molecules comprising transposons, the integration of the transposon sequences leading to a mutation or an insertion in at least one endogenous gene encoding PGM protein(s), which brings about a reduction of the expression of said at least one gene or the synthesis of inactive PGM proteins; and/or g) T-DNA molecules which, owing to insertion in endogenous genes encoding PGM protein, brings about a reduction of the expression of at least one gene encoding PGM protein(s) or the synthesis of inactive PGM proteins.

6. Use of one or more nucleic acid molecules as defined in Claim 2 for increasing the phosphate content in plant starch.

7. Use according to Claim 6, characterized in that the nucleic acid molecules encompass one or more of the nucleic acid molecules defined in Claim 5.

8. Use according to Claim 6 or 7, characterized in that the increase of the phosphate content in the starch leads to an increased content of phosphate in the C6 position of the starch in comparison with starch from corresponding wild-type plant cells which have not been genetically modified.

9. The use according to one of Claims 6 to 8, characterized in that the at least one phosphoglucomutase whose expression and/or activity has been reduced is a plastid phosphoglucomutase.

10. A method for increasing the phosphate content in starches from genetically modified plants in comparison with corresponding non-modified wild-type plants, where a) a plant cell is subjected to genetic modification according to one of Claims 1 to 5, b) a plant is regenerated from the or using the cell generated in accordance with a), and c) if appropriate, further plants are generated from the plant generated in accordance with step b).

1 1. A method for producing a starch with an increased phosphate content, comprising the extraction of the starch from a plant generated in accordance with Claim 10 and/or from starch-storing parts of such a plant and/or from a plant cell of such a plant and/or from propagation material of such a plant.