WO2001075130A1 - Transgenic plants with an increased methionine content - Google Patents

Abstract

Transgenic plants are described which have an increased methionine content due to the reduction of the activity of threonine synthase in cells of these plants. Furthermore, methods for the preparation of such cells are described as well as the use of nucleic acid molecules encoding threonine synthase for the preparation of the described transgenic plants.

Description

Transgenic plants with an increased methionine content

The present invention relates to transgenic plants which have an increased methionine content due to the reduction of the activity of a threonine synthase The present invention also relates to a method for increasing the methionine content in plants by reducing the activity of a threonine synthase as well as to the use of nucleic acid molecules encoding a threonine synthase or fragments thereof for producing plants with an increased methionine content

Essential ammo acids represent an indispensable component in the diet of monogastnc animals Plants represent one of the most important sources widely available to meet this demand A major problem, however, is that the composition of the ammo acids specifically in those plants dominating in food and feed is far away from meeting the nutritional requirements Amongst the ammo acids underrepresented methionine is by far the most important with respect to world nutrition beside lysine (Tabe and Higgins Trends in Plant Science 3 (1998), 282-286)

Methionine is an indispensable, nutritionally most important ammo acid in the diet of non-ruminant animals increasing the methionine content has been since many years one of the most important goals in plant breeding and a number of attempts have been followed (Tabe and Higgins, loc cit , Sun et al , In Biosynthesis and molecular regulation of ammo acids in plants, Singh, Flores, Shannon (Eds ) American Society of Plant Physiologists (1992), 208-228, Falco et al., Biotech 13 (1995), 577-586, Locke et al , Keystone Symposia on Molecular and Cellular Biology, Metabolic Engineering in Transgenic Plants, Abstract 306 (1997)) However, the success was rather limited, maximal increases were observed up to 40-fold in an Arabidopsis mutant (mtol) during certain developmental stages (Inaba et al , Plant Physiol 104 (1994), 881-887)

It is therefore still of utmost importance to engineer plants in order to increase their methionine content

Therefore, the technical problem underlying the present invention is to provide plants with an increased methionine content This problem is solved by the provision of the embodiments as characterized in the claims.

Accordingly, the present invention relates to transgenic plants comprising transgenic cells which are genetically modified and wherein the genetic modification leads to a reduction of the activity of a threonine synthase normally expressed in said cells when compared to corresponding wild-type cells and wherein the transgenic plant shows an increased methionine content when compared to corresponding wild-type plants.

It was surprisingly found that the reduction of the activity of threonine synthase in potato plants leads to a dramatic increase in the level of free methionine in the plants. Thus, with this approach it is now possible to provide plants having a high methionine content and which therefore meet the nutritional requirements and have an increased nutritional value.

The term "increased methionine content" means that the content of methionine is at least increased by a factor of 2 (2-fold), more preferably at least by a factor of 5 (5-fold), even more preferably at least by a factor of 10 (10-fold), particularly preferred at least by a factor of 20 (20-fold) when compared to the methionine content of corresponding wild- type plants. Furthermore, the term "preferably" means that the content of methionine is at least increased by a factor of 30 (30-fold), preferably at least by a factor of 40 (40- fold), more preferably at least by a factor of 50 (50-fold), even more preferably at least by a factor of 70 (70-fold), particularly preferred at least by a factor of 100 (100-fold) and most preferably at least by a factor of 200 (200-fold) when compared to the methionine content of corresponding wild-type plants. The term "increased methionine content" preferably means an increase of the content of free (i.e. unbound) methionine. In a particularly preferred embodiment this term means that also the content of bound methionine is increased. The amount of methionine can be determined, e.g., by HPLC (derivatization of amino acids with fluorescent labels, such as OPA, FMOC or PITC) or by GC/MS (i.e. derivatization of substances with TMS (tetramethylsilane) and converting them into gaseous compounds which are detected by mass spectrometry; see Example 3). In a particularly preferred embodiment the increase in methionine is αetermined on the basis of a GC/MS measurement as described in the Examples According to one alternative the measurement of the increase may be achieved by using an internal standard, i.e the relative amount of methionine is determined In this case the methionine content is preferably increased by a factor of at least 2, 5 10, 20, 30, 40, 50,

70, 100 or 200 In the Examples an increase between 2 and 240-fold was observed (see

Table 2)

Alternatively, the measurement of the increase of methionine can be achieved by using an external standard, i e. by calibrated measurements, which leads TO values reflecting the total amount of methionine (see Table 2, set III). In this case the methionine content is preferably increased by a factor of at least 4, 5, 10, 20, 30 or 40 in the Examples an increase between 4 7 and 42 4-fold was observed

It is possible to determine free as well as bound methionine. In a preferred embodiment the content of free (i.e. unbound) methionine is determined.

For the determination of the methionine content any suitable tissue of the plant can be used The choice of the tissue may depend on the plant species Preferred tissues are, e.g., seeds or leaves (maize), tuber (potato), peas or beans (leguminous plants), seeds

(oilseed rape).

Preferably, the methionine content is increased at least in leaf tissue

In a preferred embodiment the methionine content is increased in harvestable parts of agriculturally useful plants, such as fruit or starch storing parts e g the tuber of potatoes, or the starch storing parts of corn, grain cereals and rice

In a particularly preferred embodiment the increase of the methionine content is not accompanied by a reduction of the threonine content. This is in particular interesting in the case where the plant according to the invention is a potato plant and the increase of the methionine content occurs also in the tubers

In the context of the present invention the term "transgenic" means that the cells of the plants according to the invention comprise in their genome a foreign nucleic acid molecule which does not occur in not genetically modified cells of corresponding wild- type plants. The term "foreign nucleic acid molecule " in this context means that the nucleic acid molecule does not naturally occur in the corresponding plant cells, i.e. it is heterologous with respect to the plant cells, or that the nucleic acid molecule does not naturally occur in the plant cells in the specific spatial arrangement, i.e. it is linked to sequences other than those naturally linked to it, or that the nucleic acid molecule is located at a position in the genome of the plant cells at which it does not naturally occur. In a preferred embodiment the foreign nucleic acid molecule is a recombinant molecule which comprises different elements which do not naturally occur in the plant cells in their specific combination or spatial arrangement. The localization of the foreign nucleic acid molecule in the genome of the plant cell or its spatial arrangement can be verified or determined, e.g., by Southern Blot analysis.

The term "corresponding wild-type plants" means plants which were used as the starting material for the production of the transgenic plants, i.e. plants which have the same genetic information as the transgenic plant except for the genetic modification which was effected in the transgenic plant.

The term "genetically modified" means that the plant cells have been modified with respect to their genetic information due to the introduction of a foreign nucleic acid molecule and that the presence or the expression of the foreign nucleic acid molecule leads to a phenotypic change. Phenotypic change in this context preferably means a measurable change of one or more cell functions, such as enzymatic activities, or states of parameters, such as concentrations of metabolites. The genetically modified plant cells of the plants according to the invention show a reduction of the activity of a threonine synthase which endogenously occurs in the cells due to the presence and/or expression of a foreign nucleic acid molecule.

The term "threonine synthase" means a protein which catalyses the conversion of O- phospho-L-homoserine to threonine and inorganic phosphate (see Figure 1), preferably it is a protein having the enzymatic activity of a threonine synthase (EC 4.2.99.2). The term "reduction of the activity" in this context means a reduction of the expression of a gene(s) encoding threonine synthase which endogenously occur(s) in the plant cells, a reduction in the amount of threonine synthase protein in the plant cells and/or a reduction of the enzymatic activity of the threonine synthase in the plant cells. The reduction of the expression can, be determined, e.g., by measuring the amount of transcripts encoding threonine synthase, for example by Northern Blot analysis. The term "reduction" preferably means a reduction of the amount of transcripts encoding threonine synthase of at least 10%, more preferably of at least 30%, even more preferably of at least 50%, particularly preferred of at least 70% and most preferably of at least 90% when compared to the amount of transcripts encoding threonine synthase of corresponding not genetically modified cells.

The reduction of the amount of threonine synthase protein in plant cells can be determined, e.g., by Western Blot analysis. The term "reduction" in this context preferably means a reduction of the amount of threonine synthase protein of at least 10%, more preferably of at least 30%, even more preferably of at least 50%, particularly preferred of at least 70% and most preferably of at least 90% when compared to the amount of threonine synthase protein in corresponding not genetically modified plant cells.

The enzymatic activity of the threonine synthase can be determined as described, for example, in Curien et al. (Biochem. 31 (1998), 13212-13221) or in Laber et al. (Eur. J. Biochem. 263 (1999), 212-221 ). Moreover, the following two approaches can be used to determine the activity of threonine synthase:

(i) An enzyme assay can be used which is performed at room temperature by adding about 0.5 μg of purified threonine synthase to a solution of 100 mM tricine pH 8.5, 100 μM SAM, 50 μM PLP (pyridoxalphosphate), and 250 μM phosphohomoserine (HserP) in a total volume of 100 ml. After 15 min the reaction is terminated by addition of malachite green reagent solution and the inorganic phosphate liberated from HserP is quantified by the method of Lanzetta et al. (Anal. Biochem. 100 (1979), 95-97). Enzyme activities are expressed as nmol Pj formed per second per mg of protein.

(ii) Threonine synthase activity can be measured in a volume of 100 μl containing 100 mM Na-HEPES, pH 8.0, 1 mM HserP, 10 mM NaF, 50 μM PLP, in the absence or presence of 200 μM SAM. Assays are initiated by adding enzyme. After incubation at 30°C for 5-60 min, reactions are stopped by addition of 50 μl of 20% (w/v) TCA and the precipitated proteins are removed by centrifugation. Threonine formation is determined by high performance liquid chromatography (HPLC) after derivatization of 2-29 μl of the TCA supematants with O- phthaldialdehyd.

The term "reduction" in this context preferably means a reduction of the threonine synthase activity of at least 10%, preferably of at least 30%, more preferably of at least 50%, even more preferably of at least 70% and most preferably of at least 90% when compared to the threonine synthase activity in corresponding not genetically modified plant cells. However, it is preferred that the threonine synthase activity is not completely abolished (i.e. a reduction of 100%) since this might be lethal for the plants. Accordingly, it is preferred that the transgenic plants show a low level of threonine synthase activity, e.g. at least 5%, more preferably at least 6%, even more preferably at least 10% and particularly preferred at least 20% of the threonine synthase activity of corresponding non-genetically modified plants/plant cells.

The genetic modification of the plant cells with a foreign nucleic acid molecule the presence and/or expression of which leads to a reduction of the activity of a threonine synthase in the plant cells, can be of different kinds.

For example, the genetic modification can be the introduction of a foreign nucleic acid molecule into an endogenous threonine synthase gene wherein this introduction leads to the reduction or total repression of the expression of this gene. The term "reduction" in this context preferably means a reduction of transcription of at least 10%, preferably of at least 30%, more preferably of at least 50%, even more preferably of at least 70% and most preferably of at least 90% when compared to transcription in corresponding not genetically modified plant cells. The level of transcription can be determined, e.g., by Northern Blot analysis. Preferably, the reduction of the transcription leads to a corresponding reduction of the activity of the threonine synthase. Examples for such genetic modifications are insertions (gene disruption) or deletions in the coding region or in the promoter region of a threonine synthase gene or mutations in the coding region which lead to a premature termination of the transcription (e.g. creation of a stop codon). Another example for a genetic modification is a modification which leads to the synthesis of a threonine synthase protein which, however, has a reduced enzymatic activity or which lacks enzymatic activity. This can be achieved, e.g., by mutations in the coding region of a threonine synthase gene, which lead to the loss of enzymatic function or by mutations which lead to aberrant splicing of the threonine synthase mRNA. Methods for effecting genetic modifications in genes or their regulatory regions which lead to a reduction or total repression of expression of the gene are well known to the person skilled in the art and include, e.g., transposon mutagenesis and gene tagging. It is also conceivable to effect a genetic modification which consists in the introduction of a foreign nucleic acid molecule which codes for an antagonist/inhibitor of threonine synthase, such as an antibody the binding of which to threonine synthase leads to a reduction of its enzymatic activity.

In a preferred embodiment the genetic modification comprises the introduction of a foreign nucleic acid molecule into the genome of the plant cell expression of which leads to a reduction of the expression of endogenous genes encoding threonine synthase, in particular, on the level of transcription or translation.

A suitable approach in this regard is, e.g., the approach based on RNA interference as described, for example, in Caplen et al. (Gene 252 (2000), 95-105). Meins (Plant Mol. Biol. 43 (2000), 261-273) and Levin et al. (Plant Mol. Biol. 44 (2000), 759-775). In a particularly preferred embodiment the foreign nucleic acid molecule which is introduced into the genome of the plant cell codes for a suitable antisense RNA molecule, for a suitable ribozyme or for a suitable cosuppression RNA molecule. In the antisense RNA approach a suitable DNA sequence is expressed in antisense orientation which leads to the synthesis of an antisense RNA molecule which is complementary to the transcript of a threonine synthase gene or to parts thereof. Such an antisense RNA molecule preferably has a length of at least 30 nucleotides, more preferably of at least 50 nucleotides and most preferably of at least 100 nucleotides. The antisense RNA molecules should have a high degree of homology to the endogenous threonine synthase gene which is present in the plant cell. Preferably, the homology is at least 90%, more preferably at least 95% and most preferably at least 99%. When using the ribozyme approach, an RNA molecule is synthesized in the plant cells, which specifically cleaves transcripts of threonine synthase genes. The expression of ribozymes in cells is well known to the person skilled in the art and is described, e.g., in EP-B1 0 321 201. The expression of ribozymes in plant cells is described, e.g., in Feyter et al. (Mol. Gen. Genet. 250 (1996), 329-338).

The cosuppression approach is based on the expression of a sense RNA molecule which suppresses the expression of endogenous mRNA transcripts encoding threonine synthase. The cosuppression effect is described, e.g., 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), 31 1-317) and de Borne et al. (Mol. Gen. Genet. 243 (1994), 613-621). In a particularly preferred embodiment of the present invention the reduction of the activity of threonine synthase in the plant cells is achieved by introduction of a nucleic acid molecule comprising the following elements:

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

(b) a DNA sequence the transcripts of which are, at least in part, complementary to transcripts of an endogenous threonine synthase gene, said DNA sequence being linked to the promoter in antisense orientation.

Nucleic acid molecules encoding threonine synthase enzymes and which can be used in order to carry out the genetic modifications described herein above, are known in the art. A cDNA encoding threonine synthase from A. thaliana is described, e.g., in Curien et al. (FEBS Letters 390 (1996), 85-90) and is also disclosed in GenBank Accession No.

L41666. Furthermore, a full length genomic sequence from A. thaliana encoding threonine synthase is disclosed in Bartlem et al. (Plant Physiol. 120 (1999), 1205) and is available under GenBank Accession No. AB 027 151. Nucleotide sequences encoding threonine synthase from corn, rice, soybean and wheat are disclosed, e.g., in WO

98/55601.

Furthermore, the following sequences encoding plant threonine synthase are accessible in the Gene database (www.ncbi.nlm. nih.gov/entrez/query.fcgi?db=Nucleotide) under accession numbers:

AC008017 BAC F3N23, chromosome 1 ;

AL050352 BAC F27B13, chromosome 4;

AB020042 Phaseolus vulgaris, EST clone;

AI563068 Citrullus lanatus, EST clone; and

AI437902, Glycine max., EST clone.

A cDNA sequence encoding threonine synthase from potato is disclosed, e.g., in

Casazza (Diploma thesis, February 1999, University of Milan, Italy) and is also shown in

SEQ ID NO:1.

Corresponding sequences from other organisms, in particular from other plants, can be isolated by using methods well known in the art, such as screening cDNA or genomic libraries with a suitable probe. Furthermore corresponding sequences can be isolated by a complementation assay using an E coll strain which is deficient for the respective enzymatic activity An example for such an approach is described in Buttcher et al (J Bactenol 179 (1997) 3324- 3330) A threonine synthase deficient E coll strain which may be used for such a complementation assay is the E coll mutant GIF41 (thrC 1001 thι-1 relA spoT1) which can be obtained from the E coll Genetic Stock Center

In a preferred embodiment a nucleic acid molecule is used which encodes a plant threonine synthase, or a part of such a molecule which is long enough to achieve the desired effect i e the reduction of the activity of threonine synthase In a particularly preferred embodiment the nucleic acid molecule encodes a potato threonine synthase or a part thereof and most preferably the nucleic acid molecule comprises the sequence as set forth in SEQ ID NO 1 or a part of that sequence

In a particularly preferred embodiment the transgenic plant according to the invention simultaneously comprises a nucleotide sequence encoding a storage protein which has a high content of methionine Examples for such proteins are the 10 kDa protein (Kiπhara et al , Gene 71 (1988), 359-370), the 15 kDa protein (Peterson et al , J Biol Chem. 261 (1986), 6279-6284) and 2S albumin (Khan et al , Transgenic Res 5 (1996), 179-185), Chakraborty et al , Proc Natl Acad Sci USA 97 (2000), 3724-3729) Such a nucleotide sequence may occur in the plants endogenously or it may be introduced into the plants by techniques known to the person skilled in the art The expression of a storage protein having a high content of methionine can lead to a further increase of the methionine content of the plant since free methionine is constantly removed from the reaction equilibrium Furthermore, the incorporation of methionine into proteins can reduce the level of degradation of free methionine

In another particularly preferred embodiment the transgenic plant according to the invention furthermore comprises a nucleotide sequence encoding a protein which is involved in sulfur assimilation and leads, if expressed in plants, to a higher content of, e g , cysteme and methionine Preferably, such proteins are involved in the regulation of sulfur assimilation, in particular, they may be key points of regulation Examples for such proteins are sulfur transporters (Hawkesford, J Exp Bot 51 (2000), 131-138), APS reductases (adenosine 5'phosDhosulfate reductase, Suter et al. J Biol Chem. 275

(2000), 930-936) and SATs (serine acetyltransferase, Harms et al , Plant J. 22 (2000),

335-343.)

Such a nucleotide sequence may occur in the plant endogenously or it may be introduced into the plants by techniques known to the person skilled in the art

In general, substantially all cells of a transgenic plant according to the invention show the described genetic modification However, it is not absolutely necessary that all cells of the plants display a reduction of the activity of threonine synthase In a preferred embodiment all or substantially all cells of the plant show a reduction of the activity of threonine synthase

In another preferred embodiment only certain cells or groups of cells or certain tissues or certain organs of the plants show a reduction of the activity of threonine synthase Preferred organs are, for example, leaves, tubers, seeds or roots, in particular storage roots.

The transgenic plants may, in principle, be plants of any plant species, that is to say they may be monocotyledonous or dicotyledonous plants Preferably, the plants are useful plants cultivated by man for nutrition They are preferably starch-storing plants, for instance cereal species (rye, barley, oat, wheat, maize, millet, sago etc.), rice, pea, beans, marrow pea, cassava, potato and sweet potato, tomato, rape, soybean, hemp, flax, sunflower, sugarcane, sugar beet, cow pea or arrowroot, fiber-forming plants (e g flax, hemp, cotton), oil-storing plants (e.g. rape, sunflower, soybean) or protein-storing plants (e.g. legumes, cereals, soybeans) and vegetables. Preferably, the transgenic plants belong to the family of the Solanaceae (e.g potato, tomato, egg-plant, pepper etc ) The invention also relates to fruit trees and palms Moreover, the invention relates to forage plants (e.g. forage and pasture grasses, such as ryegrass) to alfalfa, clover and vegetable plants (e.g. tomato, lettuce, chicory). Other plants which can be used in the present invention are, e.g., manioc and coffee.

The present invention also relates to propagation material of a transgenic plant according to the invention comprising plant ceils as defined herein above. The term "propagation material" comprises those components of the plant which are suitable to produce offspring vegetatively or generatively. Suitable means for vegetative propagation are for instance cuttings, callus cultures, rhizomes or tubers. Other propagation material includes for instance fruits, seeds, seedlings, protoplasts, cell cultures etc. The preferred propagation materials are tubers and seeds. The invention also relates to harvestable parts of the plants of the invention such as, for instance, fruits, seeds, tubers, leaves, stems or rootstocks.

The present invention also relates to genetically modified plant cells, in which the genetic modification leads to a reduction of the activity of a threonine synthase normally expressed in said cells when compared to corresponding not genetically modified wild- type cells. Preferably, these cells display an increased methionine content when compared to wild-type cells.

With respect to the definitions of the various terms and the preferred embodiments, the same applies as already set forth in connection with the transgenic plants according to the invention. The plant cells according to the invention can be used to regenerate whole plants.

Furthermore, the present invention relates to a method for increasing the methionine content of plants or parts of plants by reducing the activity of threonine synthase in cells of the plants. The activity of threonine synthase may be reduced substantially in all cells of the plant or only in certain types of cells, cell groups, tissues, parts or organs of the plants.

In a preferred embodiment the reduction of the threonine synthase activity is achieved by genetically engineering a plant as described herein above. In a particularly preferred embodiment the method according to the invention comprises the following steps:

(a) genetically modifying a plant cell by introducing a foreign nucleic acid molecule, wherein said genetic modification can lead to a reduction of the activity of endogenous threonine synthase in the plant cell: and

(b) regenerating from the cell obtained according to step (a) a plant; and optionally

(c) producing further plants from the plant obtained according to step (b).

With respect to the genetic modification mentioned in step (a), the same applies as already set forth in connection with the transgenic plants according to the invention. The regeneration of plants according to step (b) of the method can be achieved by means and methods well known to the person skilled in the art

The production of further plants according to step (c) of the method can be achieved, e g , by vegetative propagation (e g by using cuttings, tubers or by callus culture and regeneration of whole plants) or by sexual reproduction Preferably, the sexual propagation is controlled l e selected plants having certain properties are crossed and propagated in a controlled manner

The present invention also relates to transgenic plants obtainable by the method according to the invention

Moreover, the present invention also relates to the use of nucleic acid molecules encoding threonine synthase or of parts of such molecules for the preparation of genetically modified plant celis or plants comprising such cells, wherein the genetic modification leads to a reduction of the activity of a threonine synthase in the plant cell or plants when compared to corresponding not genetically modified cells or plants Furthermore, the present invention relates to the use of nucleic acid molecules encoding threonine synthase or of parts of such molecules for the preparation of plants having an increased methionine content

The plants according to the invention can be prepared by any suitable technique available for the production of transgenic plants In particular, a plurality of techniques is available by which DNA can be inserted into a plant host cell These techniques include the transformation of plant cells by T-DNA using Agrobacteπum tumefaciens or Agrobacteπum rhizogenes as a transforming agent, the fusion of protoplasts, injection, electroporation of DNA, insertion of DNA by the biolistic approach and other possibilities

The use of the Agrobacteπa-mediated transformation of plant cells has been extensively investigated and sufficiently described in EP 120 516, Potrykus and Spangenberg (Eds ), In Gene transfer to plants xxn, Springer Verlag, Berlin (1995), and An et al , EMBO J 4 (1985), 277-287 Regarding the transformation of potatoes see for instance Rocha-Sosa et al (EMBO J 8 (1989), 29-33)

The transformation of monocotyledonous plants by means of Agrobacteπum-based vectors 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 Dormisse, Int. J. Plant Sci. 153 (1992), 550-555; Ritchie et al., Transgenic Res. 2 (1993), 252-265). An alternative system for transforming monocotyledonous plants is the transformation by 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, electroporation of partially permeabilized cells, insertion of DNA via glass fibers. The transformation of maize in particular has been repeatedly described in the literature (see for instance WO 95/06128, EP 0 513 849, EP 0 465 875, EP 29 24 35; Fromm et al, Biotechnology 8, (1990), 833-844; Gordon-Kamm et al., Plant Cell 2, (1990), 603-618; Koziel et al., Biotechnology 1 1 (1993), 194-200; Moroc et al., Theor. Appl. Genet. 80, (1990), 721- 726).

The successful transformation of other types of cereals has also been described for instance of barley (Wan and Lemaux, supra; Ritala et al., supra, Krens et al., Nature 296 (1982), 72-74) and wheat (Nehra et al., Plant J. 5 (1994), 285-297).

Generally, any promoter active in plant cells is suitable to express the nucleic acid molecules in plant cells. The promoter can be chosen so that the expression in the plants of the invention occurs constitutively or only in a particular tissue, at a particular time of plant development or at a time determined by external influences. The promoter may be homologous or heterologous to the plant.

Suitable promoters are for instance the promoter of the 35S RNA of the Cauliflower Mosaic Virus (see for instance US-A-5,352,605) and the ubiquitin-promoter (see for instance US-A-5, 614,399) which lend themselves to constitutive expression, the patatin gene promoter B33 (Rocha-Sosa et al., EMBO J. 8 (1989), 23-29) which lends itself to a tuber-specific expression in potatoes or a promoter ensuring expression in photosynthetically active tissues only, for instance 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 instance US-A-5,656,496, US-A- 5,639,952, Bansal et al., Proc. Natl. Acad. Sci. USA 89 (1992), 3654-3658) and the Rubisco SSU promoter (see for instance US-A-5, 034, 322; US-A-4,962,028) or the gl ute n promoter from wheat which lends itself to endosperm-specific expression (HMW promoter) (Anderson, Theoretical and Applied Genetics 96 (1998) 568-576 Thomas, Plant Cell 2, (1990), 1171-1180) the glutehn promoter from rice (Takaiwa, Plant Mol Biol 30 (1996), 1207-1221 , Yoshihara FEBS Lett 383 (1996), 213-218, Yoshihara, Plant and Cell Physiology 37 (1996) 107-111 ) the shrunken promoter from maize (Maas, EMBO J 8 (1990), 3447-3452, Werr Mol Gen Genet 202 (1986), 471-475, err, Mol Gen Genet 212 (1988) 342-350), the USP promoter the phaseohn promoter (Sengupta-Gopalan, Proc Natl Acad Sci USA 82 (1985), 3320-3324, Bustos, Plant Cell 1 (1989), 839-853) or promoters of zem genes from maize (Pedersen et al , Cell 29 (1982), 1015-1026, Quatroccio et al Plant Mol Biol 15 (1990), 81-93) H owever, promoters which are only activated at a point in time determined by external influences can also be used (see for instance WO 93/07279) In this connection, promoters of heat shock proteins which permit simple induction may be of particular interest Moreover, seed-specific promoters such as the USP promoter from Vicia faba which ensures a seed-specific expression in Vicia faba and other plants may be used (Fiedler et al , Plant Mol Biol 22 (1993), 669-679 Baumlein et al , Mol Gen Genet 225 (1991), 459-467) Moreover, fruit-specific promoters, such as described in WO 91/01373 may be used too

Moreover, a termination sequence may be present, which serves to terminate transcription correctly and to add a poly-A-tail to the transcript, which is believed to have a function in the stabilization of the transcripts Such elements are described in the literature (see for instance Gielen et al , EMBO J 8 (1989), 23-29) and can be replaced at will

These and other embodiments are disclosed and obvious to a skilled person and embraced by the description and the examples of the present invention Additional literature regarding one of the above-mentioned methods, means and applications, which can be used within the meaning of the present invention, can be obtained from the state of the art, for instance from public libraries for instance by the use of electronic means This purpose can be served inter alia by public databases, su ch as the "medlme", which are accessible via internet, for instance under the address http //www ncbi nlm nih gov/PubMed/medline html Other databases and add resses are known to a skilled person and can be obtained from the internet, for instance under the address http //www lycos com An overview of sources and information regarding patents and patent applications in biotechnology is contained in Berks TIBTECH 12 (1994), 352-364

The disclosure content of all documents cited in this application is herewith incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference

Description of the Figures

Fig. 1 shows the simplified biosynthetic pathway of the aspartate derived am o acids, especially depicting the branchpoint between threonine and methionine synthesis

Fig. 2 shows the increase of methionine in leaves of transgenic potato plants due to antisense inhibition of the expression of threonine synthase

A) The increase of methionine in leaves of potato (cv Desiree) of four independent a-StTS (threonine synthase antisense) transformants compared to the methionine level of wiidtype (wt) is given as the ratio of methionine content of transgenic compared to wiidtype plants

The plants have been cultivated for 8 weeks in the greenhouse The level of methionine has been measured as a relative response to an internal standard (ribitol) Data are mean values of each line

B) The 5 weeks old a-StTS antisense plants 16, 45, 35, 61 exhibited a slightly altered phenotype compared to the untransformed wiidtype plant In the most affected line 61 the plant height is reduced and leaf morphology is changed The other lines, though also exhibiting an increased content of methionine, show some light green areas of the leaves

C) Northern blot analysis of leaf samples reveal the StTS transcript with a size of 1 7 kb in the wiidtype plants This signal is absent in the transformed lines The hybridization signal with a size of 1 4 kb in the transgenic lines results from the transcription of the truncated a-StTS construct. Potato StTS cDNA was used as a probe.

Especially plants 35 and 45 are of high agricultural interest as they show a high increase of methionine without deleterious phenotypic effects.

Fig. 3 shows phenotype, RNA blot analysis and threonine synthase activity of TS antisense compared to wiidtype potato plants.

A: Phenotype of transgenic lines (16, 45, 35, 61) and control plants (wt). Plant 16 displays essentially a wiidtype appearance while lines 45 and 35 display weak symptoms as slight chlorosis along nleaf nervature, whereas line 61 shows growth retardation, leaf chlorosis and alterations in leaf morphology. B: TS antisense inhibition affects tuber development as with increasing phenotypical alteration of the green matter a reduction in tuber yield due to reduction in size and number of tubers is observed.

C: RNA blot analysis of transgenic and wiidtype plants for the TS-transcript. The size of the sense-transcript is 1.7 kb (upper arrow), the antisense-transcript has a length of 1.4 kb (lower arrow). Wiidtype plants (wt) only show the presence of the sense-RNA of TS. In transgenic plants 45, 35 and 61 the presence of the antisense RNA is detected while the sense messenger is absent. In case of plant 16, both the antisense and the sense RNA are visible.

D: Determination of TS activity. ¹⁴C labled OPHS was used as substrate to determine the enzyme activity of TS in wiidtype and transgenic plants in the presence of the inductor SAM (white bars) and in its absence (black bars). Standard deviations of 3 samples per line and determination are indicated as error bars and the mean value of the activities is given on top of the respective bars.

Fig. 4 shows determination of leaf metabolite compositions in TS antisense plants.

Polar meaboiites were extracted from source leaves of 8 weeks old control plants (wt) and the TS antisense transgenic lines, 5 samples each. Metabolites were determined using GC/MS. Actual concentrations of methionine, threonine, aspartic acid, isoleucine, lysine, and glutamic acid were determined using external standards for calibration. Statistically significant changes (P<0.05) are identified with an asteriks.

Fig. 5 shows the determination of tuber metabolite compositions in TS antisense plants. Polar meabolites were extracted from sink tuber parenchyma tissue of 8 weeks old control plants (wt) and the TS antisense transgenic lines, 5 samples each. Metabolites were determined using GC/MS. Actual concentrations of methionine, threonine, aspartic acid, lysine, isoleucine, and glutamic acid were determined using external standards for calibration. Statistically significant changes (P<0.05) are identified with a star.

Fig. 6 shows the analysis of methionine pathway related genes in TS antisense plants. A: Leaf RNA of 8 weeks old control plants (wt) and transgenic plant lines 16, 45, 35, and 61 was extracted and in an RNA blot experiment hybridised to cDNA probes of potato cystathionine-gamma-synthase (StCgS), cystathionine-beta- lyase (StCbL), and methionine synthase (StMS). Isocitrate dehydrogenase of potato (ICDH) was used as positive control of expected constitutive expression. B: Protein extracts of similar plant samples as for A were subjected to a protein blot analysis using polyclonal antibodies generated against cystathionine-gamma- synthase (StCgS), cystathionine-beta-lyase (StCbL), and methionine synthase (StMS). The arrow indicates the position of the respective band which's identity has been determined in pre-experiments.

C: The activity of cystathionine-gamma-synthase of leaf extracts of wiidtype plants (wt) and transgenic lines was determined by supplying OPHS and cysteine as substrates and determining the product, cystathionine, by HPLC.

The following Examples serve to further illustrate the invention.

Example 1 : Transformation of plants with a threonine synthase antisense construct

Nucleic acids manipulation and plant transformation. The cDNA encoding threonine synthase from S. tuberosum cv. Desiree (Casazza et al., Plant Sci. 157 (2000), 43-50; Casazza, Diploma thesis, February 1999, University of Milan, Italy; SEQ ID NO: 1 ) was cut from pBlueskript SK^" as a truncated Asp718/Xbal fragment and cloned in its reverse orientation with respect to the promoter into vector pBinAR-Kan (Hδfgen and Willmitzer, Plant Sci. 66 (1990), 221-230), previously cut with Asp718/Xbal. Antisense RNA expression was under the control of the cauliflower mosaic virus (CaMV) 35S promoter and terminated by the ocs element. This construct was used to transform S. tuberosum by Agrobacterium tumefaciens mediated gene transfer (Rocha-Sosa et al., EMBO J. 8 (1989), 23-29) using the strain C58C1/pGV2260 for plant transformation (Deblaere et al., Nucl. Acid Res. 13 (1985), 4777-4788). Transgenic plants were selected on kanamycin (10 mg/l), threonine (35 mg/l), and casein hydrolysate (200 mg/l) containing medium (Dietze et al., in Gene Transfer to Plants xxii (Potrykus, I. and Spangenberg, G., eds). Berlin: Springer-Verlag (1995), 24-29). The resulting transgenic plants were planted into soil and grown in the greenhouse under a 16 hours light, 8 hours dark regime at 20°C.

Example 2: RNA analysis

(a) Plant material was harvested in the morning from transformed potato plants and from wild-type plants grown in the greenhouse and was immediately frozen in liquid nitrogen prior to storage at -80°C. Northern blot analysis was performed (Hesse et al., Amino Acids 16 (1998), 113-131 ) with 40 μg of total leaf RNA (Logemann et al., Anal. Biochem. 163 (1987), 16-20) per lane. Threonine synthase transcript levels were detected by using a radioactively labelled fragment of the threonine synthase cDNA of S. tuberosum (Hesse et al., Amino Acids 16 (1998), 113-131 ). The results are shown in Figure 2.

(b) In a more extensive analysis sixty independent transgenic plant lines were regenerated and selected on the basis of reduced TS steady state mRNA levels (data not shown). Four lines representing representative examples of weak (16), medium (45, 35), and strong (61 ) inhibition were chosen for detailed analysis. For this purpose total RNA of potato leaves was prepared according to Logemann et al. (loc. cit.). Per lane 40 μg of total RNA were separated on a denaturing agarose gel (1.2%) containing 15% formaldehyde and blotted to nylon membranes being hybridized under stringent conditions with specific radioiabelled cDNA-probes (StTS: fullength cDNA fragment (Casazza et al., loc. cit.) and exposed to X-ray films. All transgenic plants (lines 16, 45, 35, 61 ) expressed the truncated antisense transcript which was 300 nucleotides shorter than the sense transcript (Figure 3C). The mRNA steady state transcript for TS is clearly detectable in leaf tissue of wiidtype plants but not in three of the transgenic lines (45, 35, 61 ) showing only the antisense RNA signal, whereas in line 16. both, a reduced hybridisation signal of the mRNA is still detectable together with the antisense RNA signal which might be due to a moderate antisense reduction or for patchy, non-uniform expression of the antisense-RNA (Hofgen et al.. Proc. Natl. Acad. Sci. USA 91 (1994), 1726-1730).

Example 3: Metabolite analysis (1)

Leaf discs (approximately 250 mg) were taken from 8 week old greenhouse grown plants and stored at -80°C. Leaf tissues were ground to a fine powder in liquid nitrogen in a bead-mill. Methanol (1400 μl), ribitol (50 μl; 0.2 mg/ml as an internal standard), and ddH₂0 (50 μl) were added to each sample. All further steps were done according to Keller et al. (Plant J. 16 (1998), 403-410).

Metabolite analysis was carried out on a GC-MS system consisting of a GC 8000 Top gas chromatograph, a Voyager quadrupole mass spectrometer (both Thermoquest, Engelsbach, Germany), and a Capillary Column SPB™-50 (30 m, 25 mm ID, 25 μm film thickness; Supelco/Sigma-Aldrich, Deisenhofen, Germany). Metabolites were identified initially by comparison with a 'find-target' library running under Finnigan MassLab software (Version 1.4V). The results of the experiments are shown in Figure 2. The following Table shows the determination of amino acid content in threonine synthase antisense plants compared to wiidtype plants (WT).

Four lines representing representative examples of weak (16), medium (45, 35) and strong (61 ) inhibition were chosen for detailed analysis. Threonine synthase antisense repression of the endogenous threonine synthase results in increased levels of methionine. Threonine is slightly decreased and lysine levels are unchanged fluctuating around the mean value.

Table 1

Example 4: Macroscopic alterations

Greenhouse grown plant material was scored for macroscopic alterations of the phenotype (Fig. 3A). Transgenic line 16 was phenotypically indistinguishable from wild- type plants, lines 45 and 35 exhibited only marginal as slight growth retardation and mild chlorosis along leaf nervature, whereas line 61 represents the subset of regenerants showing a strongly impaired phenotype exhibiting severe growth retardation, phenotypical abnormalities and massive chlorosis of leaves accompanied by a severe reduction in tuber yield due to reduced size and amount of tubers (Figure 3B).

Example 5: GC/MS analysis

Moreover, the metabolite composition of the transgenic versus wild-type plants was screened for changes associated with carbohydrate metabolism. Leaf extracts were subjected to GC/MS analysis (Roessner et al., Plant J. 23 (2000), 1-12). For this purpose leaf material (about 250 mg each) was harvested from greenhouse grown plants after approximately 8 weeks cultivation, at the beginning of floweπng period. Leaf discs have been excised from tissues of similar developmental stages. Tuber samples (about 120 mg) were taken from tubers of 5 months old plants. Sampling took place between 10 and 12 hours a.m. and plant material was immediately frozen in liquid nitrogen prior to storage at -80°C. All metabolites were determined using GC/MS based technology. Leaf tissues were ground to a fine powder in liquid nitrogen in a bead-mill. Methanol (1400 μl), ribitol (50 μl; 0.2 mg/ml as an internal standard), and ddH₂0 (50 μl) were added to each sample and successive methanol and chloroform (750 μl) extractions were performed (Maiman et al., Plant J. 23 (2000), 747-758). Tuber analysis has been performed according to previously published protocols (Roessner et al., Plant J. 23 (2000), 1-12). For the quantification of amino acids, external standards were measured in addition to the plant samples and recoveries were determined prior to analysis.

Example 6: Determination of remaining enzyme activity of StTS

TS measurement was adapted from Giovanelli et al. (Plant Physiol. 76 (1984), 285-292). Total protein was extracted from source leaves of 8 weeks old plants, which were immediately frozen in liquid nitrogen and stored at -80°C. Frozen leaf tissue was homogenized at 4°C with a micro pestle in 500 μl extraction buffer containing 50 mM HEPES (pH 7,8), 10% (v/v) glycerol, 20 μM PLP. After centrifugation at 4°C (15000 g, 20 min) the supernatant was desalted via pre-equilibrated NAP-5 columns. Protein concentration was determined according to Bradford (Anal. Biochem. 72 (1976), 248- 254). Threonine synthase activity of 25 μg desalted protein extract was determined in a radioactive assay where threonine formation was monitored in a scintillation counter. The enzyme was assayed in 100 mM HEPES (pH 7,8), 5% (v/v) glycerol, 250 μM PLP, 200 μM SAM, 1 μM Na₂W0 , 0,2 μM OPH in a final volume of 100 μl. Incubation was for 60 min at 30°C. The reaction was terminated by addition of 5 μl 1 N NaOH and 495μl H₂0. To separate radioactive threonine from radioactive OPHS, the mixture was incubated for 5 min with anion exchange resin (Biorad AG 1). After two centrifugation steps (15000 g, 2 min) 400 μl of the threonine containing supernatant was measured in 2 ml scintillation cocktail. Controls had either protein omitted or were incubated with heat denatured protein. Assays were performed with or without SAM to determine the activity with and without induction. To ensure that the radioactivity measured in the mixture after anion exchange chromatography resulted from threonine and not from homoserine some of the assays were analysed by thinlayer chromatography. 400 μl of dried assay reactions were diluted in a solution of unlabelled aminoacids (20 mM OPH, 20 mM homoserine and 20 mM threonine). Radioactive and nonradioactive components of the solution were separated (butanol: acetone: diethylamine: H₂0, 10: 10: 2: 5) and amino acids on the TLC plate were stained with ninhydrin prior to exposure to X-ray film. The enzyme activities of the transgenic lines 16, 45, 35, 61 in comparison to controls were determined in desalted plant leaf extracts using radioactively labeled [OPHS (¹⁴C)] substrate while the production of threonine was measured. L-[U- C]-homoserine (spec, activity: 463 MBq / mmol) (Amersham) was purified by preparative thinlayer chromatography (methanol : acetic acid : H₂0 = 6 : 3 : 1 ) and converted with E. coli homoserine kinase to [U- C]0PHS according to a protocol of Rognes (Threonine biosynthesis. In: Methods in Plant Biochemistry. Enzymes of primary metabolism (Dey, P.M., and Harbone, J.B. Eds.), 3 (1990), 315-324 Academic Press, New York). Phosphorylation of L-homoserine was performed at 37°C, for 4 h in 500 μl of 5 mM HEPES (pH 7.5), 1.5 mM ATP, 1.5 mM MgCI, 1 mM L-[U- C]-homoserine. The reaction was started by the addition of 5 U of homoserine kinase. The reaction was terminated by addition of 15 μl 1 N HCI and the solution was applied immediately to a cation exchange column ((Biorad AG 50W). [U- C]OPHS was eluted with H₂0. The enzyme activity was determined with and without SAM. In the presence of SAM wild-type extracts revealed a TS activity of 25.3 pmol per minute and mg protein. For the antisense lines remaining activities of 16% (16), 12% (45), 10% (35), and 6% (61), respectively, were determined (Figure 3D). Without the activator SAM the TS activity determined in wild-type leaf extracts was reduced to about 4.8 % , and in the transgenic lines (16, 45, 35, 61) to about 2.0%, 1.7%, 1.9%, and 2.3%, respectively.

Example 7: Effect of threonine synthase antisense inhibition on metabolite levels in source leaves

Leaves are supposed to be the main organ for amino acid biosynthesis in plants (Wallsgrove et al., Plant Physiol. 71 (1983), 780-784; Ravanel et al., Proc. Natl. Acad. Sci. USA (1998), 7805-7812). To address the question whether the down-regulation of TS through antisense inhibition leads to changes in amino acid contents source leaf extracts of these plants were analysed using GC/MS based technology (Roessner et al., loc. cit.; see also Example 5, supra). For a first evaluation relative responses correlated to the internal standard ribitol have been determined and the amount of threonine in the lines 16, 45, 35, and 61 was found to be reduced to 50%, 30%, 16%, and 13% of wild- type (data not shown). This is in perfect agreement with setting the block at the level of the threonine synthase. In order to score for the effect of TS inhibition on the competing methionine pathway three successive sets of plants (set I, II, III) were grown in the greenhouse and analysed. The levels of free methionine of these plants were increased in all four transgenic plant lines ranging between 2- and 240-fold at maximum (Table 2).

Table 2 Relative methione contents of TS antisense plants compared to wiidtype.

experiment Wild-type 16 45 35 61 set I 1 91 192 222 181 set II 1 2 13 53 239 set III 1 2 10 10 16

Potato plants were grown in three successive sets of experiments. Relative responses of methionine in leaf extracts of 8 weeks old plants were determined in comparison to an internal ribitol standard using GC/MS measurements. The concentration of methionine of the respective wild-type extracts (wt) was set as 1 and for extracts of transgenic TS antisense plant lines (16, 45, 35, 61) the ratio to the wild-type concentration is presented.

Probably depending on the developmental status or on the respective growth conditions, variations in the actual methionine levels of individual plants within the same experiment and between the three sets of experiments were observed. Noteworthy, even lines without substantial alteration in morphology (16, 45, 35) accumulated increased levels of methionine.

For the experimental set III a detailed metabolite analysis was performed in order to determine actual metabolite concentrations and the GC/MS measurements were thus calibrated with externally supplied standards (Figure 4). The methionine content was determined as 24, 128, 137, and 220 nM methionine /g FW (lines 16, 45, 35, and 61 , respectively) compared to the extremely low levels of free methionine in wild-type plants (5 nM/g FW). This corresponds to increases of about 44-fold at maximum for the absolute values of experimental set III, though these factors have to be considered with some caution due to the extremely low concentrations of methionine in wild-type leaf tissue. The GC/MS based analysis of further amino acids, respectively intermediates of methionine biosynthesis in source leaves, supports the interpretation of the increased methionine levels as largely resulting from the redirection of carbon flow from the threonine to the methionine branch (Figure 4). The small but significant increase observed for aspartic acid indicates the importance of threonine synthase for carbon allocation into the aspartate pathway and might hint at a regulatory mechanism signalling carbon demand for the whole pathway when threonine levels are low. Lysine and isoleucine levels did not change, essentially. The amount of cysteine, as the second necessary precursor of cystathionine biosynthesis, providing reduced sulfur, seems not to be altered in the threonine antisense plants of wild-type appearance (16, 45, 35) but in the severely affected plant (61), only. This plant (61) shows altogether a rather perturbed amino acid profile reflecting major metabolic problems resulting from threonine deficiency. The levels of glutamic acid (Figure 4) and vaiine and alanine (data not shown), amino acids not directly related to the aspartate pathway, remained unchanged. Furthermore, increasing concentrations were determined on the basis of relative responses (data not shown) of the pathway intermediates homocysteine (max of 46-fold), an intermediate which is hardly detectable in wild-type plants and the direct precursor of methionine, and homoserine (max of 175-fold).

Example 8: Effect of threonine synthase antisense inhibition on metabolite levels in tubers

Tubers are the major sink tissue of the potato crop. Therefore the parenchyma tissue of tubers of experimental set III (Table 2) was analysed for its amino acid composition using GC/MS (Roessner et al., loc. cit.). Already in wild-type plants the amount of free methionine is about one order of magnitude higher in tuber tissues (1.2 μM/g FW) than in leaf tissues of the corresponding plants (0.005 μM/g FW). In contrast to the results obtained for leaves one could not observe a reduction of threonine contents in potato tubers of the TS antisense plants but rather constant levels of free threonine of about 0.5 μg/g FW (Figure 5). Yet, the methionine content was increased up to 30-fold in tubers (Figure 5). Lines 16, 45, 35, and 61 contained 9.7, 9.8, 20.2, and 35.4 μM methionine/g FW, respectively. As in leaves, lysine contents were not altered, however, isoleucine levels were increased e.g. in line 35 by a factor of 5 to 8.3 μM/g FW. Vaiine, alanine (data not shown) and glutamate (Figure 5) as amino acids not related to the aspartate family were not altered in these tissues. Corresponding to the fact that threonine levels are not reduced in tubers other than in leaves no increase of aspartic acid was observed. Whether the observed increases are dependent on effective import processes or to what extent in situ biosynthesis adds to the accumulation of amino acids in tubers remains to be determined.

Example 9: Analysis of methionine pathway related genes in TS antisense plants

In Arabidopsis and Lemna elevated levels of methionine resulted in reduced steady state levels of CgS messenger RNA or protein contents, respectively (Inaba et al.. Plant Physiol. 104 (1994), 881-887; Thompson et al., Plant Physiol. 69 (1982), 1077-1083; Chiba et al., Science 286 (1999), 1371-1374). It was tested whether alterations in TS expression and the accompanying changes in metabolites, especially the observed endogenous increase in free methionine, would influence expression and protein content of methionine biosynthetic genes, i.e. cystathionine gamma-synthase (CgS), cystathionine beta-lyase (CbL) and methionine synthase (MS). RNA blot analysis using CgS, CbL and MS as probes revealed no effect of TS inhibition or the accompanying methionine increase on CgS, CbL or MS steady state RNA levels in transgenic potato plants compared to wild-type (Figure 6A; StCgS: 1 ,3 kb internal EcoRI fragment (Riedel et al., Plant Biol. 1 (1999), 638-644); StCbL: 1 ,3 kB BamHI/Sacl fragment, a strand specific cRNA-probe has been synthesized (Riboprobe, Promega Madison); StMS: full length cDNA (Zeh, personal communication). Isocitrate dehydrogenase (ICDH) was used as positive control showing as expected a constitutive expression (Fieuw et al., Plant Physiol. 107 (1995), 905-913). Furthermore, protein blot analysis of CgS, CbL and MS using polyclonal antibodies (Maiman et al., loc. cit.) provided no hint for changes in the protein contents of the respective enzymes (Figure 6B).

Example 10: Determination of CgS enzyme activity in TS antisense plants

Given the fact that the activity of CgS, the enzyme competing with TS for the common substrate OPHS, is of interest with respect to understanding the allocation of metabolites, the activity of CgS in desalted plant extracts was determined. Cystathionine, as the specific product of this reaction, was determined by HPLC. In particular, CgS activity was measured as described by Ravanel et al. (Arch. Biochem. Biophys. 316 (1995), 572-684). Leaf tissues (100 mg) from source-leaves of 8 week old plants were collected and immediately frozen in liquid nitrogen. The samples were then kept at -80°C until the assay was performed. Frozen leaf tissue was ground using a micro pestle in 500 μl ice-cold extraction buffer containing 20 mM MOPS-NaOH (pH 7.5), 2 mM DTT, 100 μM PLP, 0.1 % (v/v) Triton X, 1 mM EDTA, 0.2% (w/v) PMSF. After two centrifugation steps (14 000 g, 15 min, 4°C) the supernatant was desalted by using pre-equilibrated NAP-5 columns (Pharmacia). CgS activity was measured in a volume of 100 μl containing 20 mM MOPS-NaOH (pH 7.5), 2 mM DTT, 0.1 mM PLP, 2 mM L- cysteine, 5 mM O-phospho-L-homoserine and 0.2 mM L-α-(aminoethoxyvinyl)glycine (AVG). AVG is known to act as a specific inhibitor for cystathionine β-lyase (Droux et al., Arch. Biochem. Biophys. 316 (1995), 585-595), the enzyme catalysing the subsequent step in methionine biosynthesis in plants, and was added in order to prevent enzymatically formed L-cystathionine from being further converted to L-homocysteine. Assays were initiated by adding the desalted protein extract (100 μg). After incubating the mixture for 60 min at 30°C the reaction was stopped by boiling for 5 min and L- cystathionine formation was analysed by high-performance liquid chromatography (HPLC) after derivatisation with O-phthaldialdehyde (OPA).

The enzymatic activity of wild-type leaf extracts was determined as 0.68 nmol cystathionine per minute and mg total protein. The activities determined for the transgenic lines (16, 45, 35, and 61 ) were 0.62, 0.60, 1.0 and 1.2 nmol min-1 ιτιg-1 , respectively (Figure 6C). Although this might indicate a slight increase of CgS activity with reducing TS activity, the increase was statistically not significant, especially due to higher variations in the plants more strongly inhibited and showing aberrant phenotypes. We therefore assume that the activity of CgS remains, at least, constant when TS activity is reduced and methionine levels are increased.

Claims

1. A transgenic plant comprising transgenic plant cells, which are genetically modified and wherein the genetic modification leads to a reduction of the activity of a threonine synthase normally expressed in said cells when compared to corresponding wild-type cells and wherein said transgenic plant shows an increased methionine content when compared to corresponding wild-type plants.

2. The transgenic plant of claim 1 , wherein the genetic modification comprises the introduction of a foreign nucleic acid molecule into the genome of the plant cells contained in the plant and wherein the expression and/or presence of said foreign nucleic acid molecule leads to the inhibition of the expression of endogenous threonine synthase genes present in the genome of the plant cells.

3. The transgenic plant of claim 2, wherein the foreign nucieic acid molecule is selected from the group consisting of

(a) a nucleic acid molecule encoding an antisense RNA molecule which is, at least in part, complementary to transcripts of a threonine synthase gene;

(b) a nucleic acid molecule encoding a ribozyme which specifically cleaves transcripts of a threonine synthase gene; and

(c) a nucleic acid molecule which encodes a cosuppression RNA molecule (sense RNA molecule) which corresponds to a transcript of a threonine gene.

4. The transgenic plant of any one of claims 1 to 3, wherein the activity of the threonine synthase is reduced in cells of substantially all parts of the plant.

5. The transgenic plant of any one of claims 1 to 4, wherein the activity of the threonine synthase is reduced by at least 10% when compared to the activity in corresponding not genetically modified wild-type cells.

6. The transgenic plant of any one of claims 1 to 5, wherein the methionine content is increased by at least 5-fold when compared to the methionine content of corresponding not genetically modified wild-type plants.

7. The transgenic plant of any one of claims 1 to 6, which furthermore comprises a nucleotide sequence encoding a storage protein which has a high content of methionine.

8. The transgenic plant of any one of claims 1 to 7, which furthermore comprises a nucleotide sequence encoding a protein which is involved in sulfur assimilation.

9. The transgenic plant of any one of claims 1 to 8 which is selected from the group consisting of maize, wheat, barley, sugar beet, sugar cane, rape, vegetables, soybean, bean, pea, rice, potato or sweet potato, tomato, egg-plant, pepper, cassava or manioc.

10. The transgenic plant of claim 9, which is a potato plant and which shows an increase in the methionine content in the tubers.

11. The transgenic potato plant of claim 10, which does not show a reduction of the threonine content in the tubers.

12. Propagation mateπal of the plant of any one of claims 1 to 11 comprising genetically modified plant cells as defined in claim 1.

13. A tuber of a potato plant of claim 10 or 11 comprising genetically modified plant cells as defined in claim 1.

14. A method for increasing the methionine content in plants compπsing the step of reducing the activity of threonine synthase in cells of the plants.

15. The method of claim 14, which comprises the steps of (a) genetically modifying a plant cell by introducing a foreign nucleic acid molecule, wherein said genetic modification can lead to a reduction of the activity of endogenous threonine synthase in the plant cell; and

(b) regenerating from the cell obtained according to step (a) a plant; and optionally

(c) producing further plants from the plant obtained according to step (b).

16. Use of a nucleic acid molecule encoding threonine synthase or of parts of such a molecule for the preparation of genetically modified plant cells or plants comprising such cells, wherein the genetic modification leads to a reduction of the activity of a threonine synthase in the plant cells when compared to corresponding not genetically modified cells.

17. Use of a nucleic acid molecule encoding threonine synthase or of parts of such a molecule for the preparation of plants having an increased methionine content.