AU757440B2 - DNA sequence coding for a 1-deoxy-d-xylulose-5-phosphate synthase and the overproduction thereof in plants - Google Patents

Description

DNA sequence coding for a 1-deoxy-D-xylulose-5-phosphate synthase and overproduction thereof in plants The present invention relates to the use of DNA sequences coding for a 1-deoxy-D-xylulose-5-phosphate synthase (DOXS) for producing plants with increased tocopherol, yitamin

K,

chlorophyll and/or carotenoid contents., ;preferably to the use of a DNA sequence SEQ ID No. 1 or SEQ ID No. 3 or of a DNA sequence hybridizing with the latter, to the use of a DNA sequence SEQ ID No. 1 or SEQ ID No. 3 and of a DNA sequence SEQ ID No. 5 or DNA sequences hybridizing with the latter and coding for a 1-deoxy-D-xylulose-5-phosphate synthase (DOXS) and a p-dydroxyphenylpyruvate dioxygenase (HPPD) for producing plants with increased content of tocopherols, vitamin K, chlorophylls and/or carotenoids, to the use of a DNA sequence SEQ ID No. 1 or SEQ ID No. 3 and of a DNA sequence SEQ ID No. 7 or DNA sequences hybridizing with the latter and coding for a synthase (DOXS) and a 20 geranylgeranyl-pyrophosphate oxidoreductase (GGPPOR) for producing plants with increased content of tocopherols, vitamin K, chlorophylls and/or carotenoids, to the use of a DNA sequence SEQ ID No. 1 or SEQ ID No. 3, of a DNA sequence SEQ ID No. 5 and of a DNA sequence SEQ ID No. 7 or DNA sequences hybridizing with the latter and coding for a synthase (DOXS), a hydroxyphenylpyruvate dioxygenase (HPPD) and a geranylgeranyl-pyrophosphate oxidoreductase (GGPPOR) for producing plants with increased content of tocopherols, vitamin K, chlorophylls and/or carotenoids, to processes for producing 30 plants with increased tocopherol, vitamin K, chlorophyll and/or carotenoid contents, comprising a DNA sequence SEQ-ID No. 1 or SEQ ID No. 3; SEQ ID No. 1 or SEQ ID No. 3 and SEQ ID No. 5; SEQ ID No. 1 or SEQ ID No. 3 and SEQ ID No. 7 or a DNA sequence SEQ ID No. 1 or SEQ ID No. 3 and SEQ ID No. 5 and SEQ ID No. 7, to 35 the plants themselves produced in this way, and to the use of SEQ ID No. 1 or SEQ ID No. 3 for producing a test system for identifying DOXS inhibitors.

An important aim of molecular genetic work on plants to date has been the generation of plants with increased content of sugars, enzymes and amino acids. However, there is also commercial interest in the development of plants with increased content of vitamins, such as increasing the tocopherol content.

S= 5 The eight compounds with vitamin E activity which occur in nature a are derivatives of 6-chromanol (Ullmann's Encyclopedia of Industrial Chemistry, Vol. A 27 (1996), VCH Verlagsgesellschaft, 0817/00006 2 Chapter 4, 478-488, Vitamin The first group (la-d) is derived from tocopherol, while the second group consists of derivatives of tocotrienol (2a- d): R1

HO

R 4' 8' R3 la, a-Tocopherol: R 1

R

2

R

3

CH

3 lb, P-Tocopherol [148-03-8]: R 1

R

3

CH

3

R

2

H

Ic, y-Tocopherol [54-28-4]: R 1 H, R 2

R

3

CH

3 Id, 6-Tocopherol [119-13-1]: R 1

R

2 H, R 3

CH

3 R1

HO

R 3' 7' 11' R3 2a, a-Tocotrienol [1721-51-3]: R 1

R

2

R

3

CH

3 2b, (-Tocotrienol [490-23-3]: R 1

R

3

CH

3

R

2

H

2c, y-Tocotrienol [14101-61-2]: R 1 H, R 2

R

3

CH

3 2d, 6-Tocotrienol [25612-59-3]: R 1

R

2 H, R 3

CH

3 The compound of great commercial importance is a-tocopherol.

There are limits on the development of crop plants with increased tocopherol content through tissue culture or seed mutagenesis and natural selection. Thus, on the one hand, the tocopherol content must be measurable even in the tissue culture and, on the other hand, the only plants which can be manipulated by tissue culture techniques are those which can be regenerated to whole plants from cell cultures. In addition, crop plants may, after mutagenesis and selection, show unwanted properties which must be eliminated again by backcrossings, several times in some instances. Moreover increasing the tocopherol content by crossing would be retricted to plants of the same species.

For these reasons, the genetic engineering procedure of isolating, and transferring into target crop plants, an essential biosynthesis gene coding for tocopherol synthesis activity is 0817/00006 3 superior to the classical breeding method. The preconditions for this process are that the biosynthesis and its regulation are known and that genes which influence the biosynthetic activity are identified.

Isoprenoids or terpenoids consist of various classes of lipid-soluble molecules and are formed partly or completely of

C

5 -isoprene units. Pure prenyl lipids carotenoids) consist of C skeletons derived exclusively from isoprene units, whereas mixed prenyl lipids chlorophyll) have an isoprenoid side chain connected to an aromatic nucleus.

The biosynthesis of prenyl lipids starts from 3 x acetyl-CoA units, which are converted via B-hydroxymethylglutaryl-CoA (HMG-CoA) and mevalonate into the initial isoprene unit (C 5 isopentenyl pyrophosphate (IPP). It has recently been shown by in vivo feeding experiments with C 13 that a mevalonate-independent pathway is followed in various eubacteria, green algae and plant chloroplasts to produce IPP: 0817/00006 4 Glyceraldehyde 3-phosphate pyruvate

DOXS

1-deoxy-D-xylulose DMAP

IPP

IPP

GPP

2 IPP GGPP--- carotenoids

PPP

tocopherols This entails hydroxyethylthiamine, which is produced by decarboxylation of pyruvate, and glyceraldehyde 3-phosphate (3-GAP) being converted, in a "transketolase" reaction mediated by l-deoxy-D-xylulose-5-phosphate synthase, initially into (Schwender et al., FEBS Lett.

414(1),129-134(1997); Arigoni et al., Proc.Natl.Acad.Sci USA 94(2), 10600-10605 (1997); Lange et al., Proc.Natl.Acad.Sci.USA 95(5), 2100-2104(1998); Lichtenthaler et al., FEBS Lett. 400(3), 271-274(1997). The latter is then converted by an intramolecular rearrangement into IPP (Arigoni et al., 1997). Biochemical data indicate that the mevalonate pathway operates in the cytosol and leads to the production of phytosterols. The antibiotic mevinolin, a specific inhibitor of mevalonate production, leads only to inhibition of sterol biosynthesis in the cytoplasm, whereas prenyl lipid production in the plastids is unaffected (Bach and Lichtenthaler, Physiol. Plant 59(1983), 50-60. By S0817/00006 contrast, the mevalonate-independent pathway has a plastidic localization and leads mainly to the production of carotenoids and plastidic prenyl lipids (Schwender et al., 1997; Arigoni et al, 1997).

IPP is in equilibrium with its isomer, dimethylallyl pyrophosphate (DMAPP). Condensation of IPP with DMAPP in a head-tail addition affords the monoterpene (Cio) geranyl pyrophosphate (GPP). Addition of further IPP units results in the sesquiterpene

(C

15 farnesy pyrophosphate (FPP) and the diterpene (C 20 geranyl-geranyl pyrophosphate (GGPP). Linkage of two GGPP molecules results in the production of the C 40 precursors for carotenoids. GGPP is transformed by a prenyl chain hydrogenase into phytyl pyrophosphate (PPP), the starting material for further production of tocopherols.

The ring structures of the mixed prenyl lipids which lead to the production of vitamins E and K comprise quinones whose initial metabolites are derived from the shikimate pathway. The aromatic amino acids phenylalanine and tyrosine are converted into hydroxyphenylpyruvate, which is transformed by dioxygenation into homogentisic acid. The latter is bound to PPP in order to produce 2-methyl-6-phytylquinol, the precursor of a-tocopherol and a-tocoquinone. Methylation steps with S-adenosylmethionine as methyl group donor result initially in 2,3-dimethyl-6-phytylquinol and then, by cyclization, in y-tocopherol and, by methylation again, in a-tocopherol (Richter, Biochemie der Pflanzen, Georg Thieme Verlag Stuttgart, 1996).

Examples are to be found in the literature showing that manipulation of an enzyme can influence the direction of the metabolyte flux. It was possible in experiments with modified expression of phytoene synthase, which links two GGPP molecules together to give 15-cis-phytoene, to measure a direct effect on the amounts of carotenoids in these transgenic tomato plants (Fray and Grierson, Plant Mol.Biol.22(4),589-602(1993); Fray et al., Plant 8, 693-701(1995). As expected, transgenic tobacco plants with reduced amounts of phenylalanine-ammonium lyase show reduced phenylpropanoid amounts. The enzyme phenylalanine-ammonium lyase catalyzes the breakdown of phenylalanine and thus removes it from phenylpropanoid biosynthesis (Bate et al.,Proc. Natl. Acad. Sci USA 91 (16): 7608-7612 (1994); Howles et al., Plant Physiol. 112.

1617-1624(1996).

0 0817/00006 6 To date, little has been disclosed about increasing the metabolite flux in order to increase the tocopherol content of plants through overexpression of individual biosynthesis genes.

There is merely a description in WO 97/27285 of modification of the tocopherol content by increased expression or by down-regulation of the enzyme p-hydroxyphenylpyruvate dioxygenase

(HPPD).

It is an object of the present invention to develop a transgenic plant with increased content of tocopherols, vitamin K, chlorophylls and carotenoids.

We have found that this object is achieved by overexpression of a sythase (DOXS) gene in the plants.

In order to increase the metabolite flux from primary metabolism into isoprenoid metabolism, the production of IPP as general starting substrate for all plastidic isoprenoids was increased.

For this purpose, the DOXS activity in plants was increased by overexpression of the homologous gene (gene from organism of the same species). This can also be achieved by expressing a heterologous gene (gene from remote organisms). Nucleotide sequences from Arabidopsis thaliana DOXS (Acc. No. U 27099), rice (Acc. No. AF024512) and peppermint (Acc. No. AF019383) are described.

In one example 1 there is enhanced expression of the DOXS gene from Arabidopsis thaliana (SEQ ID No.:l; Mandel et al., Plant J.

9, 649-658(1996); Acc. No. U27099) in transgenic plants.

Plastidic localization is ensured by the transit signal sequence present in the gene sequence. A suitable expression cassette is also a DNA sequence which codes for a DOXS gene which hybridizes with SEQ ID No. 1 and which is derived from other organisms such as, for example, E. coli (SEQ ID No.3) or, preferably, from other plants.

The GGPP which is now available in increased quantities is converted further in the direction of tocopherols and carotenoids.

Efficient production of carotenoids is essential for photosynthesis, where they serve together with chlorophylls as "light-collecting complexes" for better utilization of the energy of photons (Heldt, Pflanzenbiochemie. Spektrum Akademischer Verlag Heidelberg Berlin Oxford, 1996). In addition, carotenoids carry out important functions protecting from oxygen free radicals such as singlet oxygen, which they are able to return to 0817/00006 7 the ground state (Asada, 1994; Demming-Adams and Adams, Trends in Plant Sciences 1; 21-26(1996). A synthase-defective Arabidopsis thaliana mutant showing an "albino phenotype" has been isolated (Mandel et al., 1996). It can be inferred from this that a reduced amount of carotenoids in the plastids has adverse effects on the plant.

We have found that the object is also achieved by overexpression of a 1-deoxy-D-xylulose-5-phosphate synthase (DOXS) gene and of a p-hydroxyphenylpyruvate dioxygenase (HPPD) gene in the plants, see Figure 1.

In order to increase the metabolite flux from primary metabolism into isoprenoid metabolism, the production of IPP as general starting substrate for all plastidic isoprenoids was increased.

For this purpose, the DOXS activity in transgenic tobacco and oilseed rape plants was increased by overexpression of the DOXS from E. coli. This can be achieved by expression of homologous or other heterologous genes.

The D-1-deoxy-xylulose 5-phosphate which is now available in increased quantities is converted further in the direction of tocopherols and carotenoids.

In addition, the production of homogentisic acid further intensifies the metabolite flux in the direction of phytylquinones and thus tocopherol, see Figure 1. Homogentisic acid is produced from p-hydroxyphenylpyruvate by the enzyme p-hydroxyphenylpyruvate dioxygenase (HPPD). cDNAs coding for this enzyme have been described from various organisms such as, for example, from microorganisms, from plants and from humans.

In Example 11 there was for the first time overexpression of the HPPD gene from Streptomyces avermitilis (Denoya et al., J. Bacteriol. 176(1994), 5312-5319; SEQ ID No. 5) together with the DOXS from E. coli SEQ ID No. 3 in plants and plant plastids.

The increase in the plastidic IPP production leads to enhanced production of all plastidic isoprenoids. The increased provision of homogentisic acid ensures that sufficient substrate is available for the production of tocopherols in the plastids. This homogentisate which is now available in increased quantities can in turn be converted in the transgenic plants with the amount, which is increased due to the overexpression of DOXS, of phytyl diphosphate (PPP). PPP occupies a key position, in this connection, because it serves on the one hand as starting 0817/00006 8 substrate for chlorophylls and phylloquinones, and on the other hand for tocopherols.

The transgenic plants are produced by transformation of the plants with a construct containing the DOXS and HPPD genes.

Tobacco and oilseed rape were employed as model plants for the production of tocopherols, vitamin K, chlorophylls and carotenoids.

The invention also relates to the use of the DNA sequences SEQ ID No. 1 or SEQ ID No. 3 and SEQ ID No. 5, which code for a DOXS or HPPD or functional equivalents thereof, for producing a plant with increased tocopherol, vitamin K, chlorophyll and/or carotenoid contents. The nucleic acid sequences may in these cases be, for example, DNA or cDNA sequences. Coding sequences suitable for insertion into an expression cassette are, for example, those coding for a DOXS or HPPD and conferring on the host the ability to overproduce tocopherol.

The expression cassettes additionally comprise regulatory nucleic acid sequences which control the expression of the coding sequence in the host cell. In a preferred embodiment, an expression cassette comprises upstream, i.e. at the 5' end of the coding sequence, a promoter and downstream, i.e. at the 3' end, a polyadenylation signal and, where appropriate, further regulatory elements which are operatively linked to the coding sequence for the DOXS or HPPD gene located in between.

An expression cassette is produced by fusing a suitable promoter to a suitable DOXS or HPPD DNA sequence and preferably a DNA which is inserted between promoter and DOXS or HPPD DNA sequence and codes for a chloroplast-specific transit peptide, and a polyadenylation signal by conventional recombination and cloning techniques as described, for example, in T. Maniatis, E.F.

Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989), and in T.J. Silhavy, M.L. Berman and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and in Ausubel, F.M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience (1987).

It is also possible to use expression cassettes whose DNA sequence codes for a DOXS or HPPD fusion protein, where part of the fusion protein is a transit peptide which controls translocation of the polypeptide. Transit peptides which are specific for chloroplasts and which are eliminated enzymatically 0817/00006 9 from the DOXS or HPPD part after translocation of the DOXS or HPPD gene into the chloroplasts are preferred. The particularly preferred transit peptide is derived from the plastidic transketolase (TK) or a functional equivalent of this transit peptide (for example the transit peptide of the small subunit of rubisco or ferredoxin-NADP oxidoreductase).

The fused expression cassette coding for a DOXS gene and an HPPD gene is preferably cloned into a vector, for example pBinl9, which is suitable for transformation of Agrobacterium tumefaciens.

The invention further relates to the use of an expression cassette comprising DNA sequences SEQ ID No. 1 or SEQ-ID No. 3 and SEQ ID No. 5 or DNA sequences hybridizing with the latter for the transformation of plants or cells, tissues or parts of plants. The preferred aim of the use is to increase the tocopherol, vitamin K, chlorophyll and carotenoid contents of the plant.

It is moreover possible, depending on the choice of the promoter, for expression to take place specifically in the leaves, in the seeds or other parts of the plant. The present invention further relates to such transgenic plants, propagation material thereof and cells, tissues or parts of these plants.

The invention additionally relates to transgenic plants transformed with an expression cassette comprising the sequence SEQ ID No. 1 or SEQ ID No. 3 and SEQ ID No. 5 or DNA sequences hybridizing with the latter, and transgenic cells, tissues, parts and propagation material of such plants. Particular preference is given in this connection to transgenic crop plants such as, for example, barley, wheat, rye, corn, oats, soybean, rice, cotton, sugarbeet, canola, sunflower, flax, hemp, potato, tobacco, tomato, oilseed rape, alfalfa, lettuce and the various tree, nut and vine species.

The invention further relates to: Process for transforming a plant, which comprises introducing expression cassettes comprising a DNA sequence SEQ ID No. 1 or SEQ ID No. 3 and a DNA sequence SEQ ID No. 5 or DNA sequences hybridizing with the latter into a plant cell, into callus tissue, a whole plant or protoplasts of plants, 0817/00006 Use of the DNA sequence SEQ ID No. 1 or SEQ ID No. 3 and SEQ ID No. 5 or DNA sequences hybridizing with the latter to produce plants with increased tocopherol, vitamin K, chlorophyll and/or carotenoid contents by expression of a DOXS and an HPPD DNA sequence in plants.

The object have also been achieved by overexpression of a synthase (DOXS) gene and of a geranylgeranyl-pyrophosphate oxidoreductase (GGPPOR) gene in the plants, see Figure 1.

In order to increase the metabolite flux from primary metabolism into isoprenoid metabolism, the production of IPP as general starting substrate for all plastidic isoprenoids was increased.

For this purpose, the DOXS activity in transgenic tobacco and oilseed rape plants was increased by overexpression of the DOXS from E. coli. This can be achieved by expression of homologous or other heterologous genes.

In order to convert the GGPP which is now available in increased quantities in the direction of tocopherols and carotenoids, in a further step essential to the invention in addition the activity of the enzyme geranylgeranyl-pyrophosphate oxidoreductase is increased by overexpression of a corresponding gene. This measure achieves an increased production of phytyl pyrophosphate through increased conversion of geranylgeranyl pyrophosphate into phytyl pyrophosphate.

This is done, for example, by enhanced expression of the GGPPOR gene from Arabidopsis thaliana (SEQ ID No. 7) in transgenic plants. In order to ensure plastidic localization, a transit signal sequence is put in front of the Arabidopsis GGPPOR. Also suitable as expression cassette is a DNA sequence coding for a GGPPOR gene which hybridizes with SEQ ID No. 7 and which is derived from other organisms or from other plants.

Example 15 describes the cloning of the GGPPOR gene from Arabidopsis thaliana.

Increasing the plastidic 1-deoxy-D-xylulose 5-phosphate and phytyl pyrophosphate production leads to increased production of all plastidic isoprenoids, so that sufficient substrate for the production of tocopherols, chlorophylls, vitamin K and phylloquinones is available in the plastids.

0817/00006 11 The transgenic plants are produced by transformation of the plants with a construct containing the DOXS and GGPPOR genes.

Tobacco and oilseed rape were employed as model plants for the production of tocopherols, vitamin K, chlorophylls and carotenoids.

The invention also relates to the use of the DNA sequences SEQ ID No. 1 or SEQ ID No. 3 and SEQ ID No. 7, which code for a DOXS or GGPPOR or functional equivalents thereof, for producing plants with increased tocopherol, vitamin K, chlorophyll and/or carotenoid contents. The nucleic acid sequences may in these cases be, for example, DNA or cDNA sequences. Coding sequences suitable for insertion into an expression cassette are, for example, those coding for a DOXS or GGPPOR and conferring on the host the ability to overproduce tocopherol.

The expression cassettes additionally comprise regulatory nucleic acid sequences which control the expression of the coding sequence in the host cell. In a preferred embodiment, an expression cassette comprises upstream, i.e. at the 5' end of the coding sequence, a promoter and downstream, i.e. at the 3' end, a polyadenylation signal and, where appropriate, further regulatory elements which are operatively linked to the coding sequence for the DOXS or GGPPOR gene located in between. Operative linkage means sequential arrangement of promoter, coding sequence, terminator and, where appropriate, further regulatory elements in such a way that each of the regulatory elements can properly carry out its function in the expression of the coding sequence.

The sequences which are preferred for the operative linkage, but are not restricted thereto, are targeting sequences to ensure subcellular localization in the apoplast, in the vacuole, in plastids, in the mitochondrion, in the endoplasmic reticulum in the cell nucleus, in elaioplasts or other compartments and translation enhancers such as the 5' leader sequence from tobacco mosaic virus (Gallie et al., Nucl. Acids Res. 15 (1987), 8693-8711).

For example, the plant expression cassette can be incorporated into the tobacco transformation vector pBinAR-Hyg. Fig. 1 shows the tobacco transformation vectors pBinAR-Hyg with the promoter and pBinAR-Hyg with the seed-specific promoter phaseolin 796 HPT: hygromycin phosphotransferase OCS: octopine synthase terminator PNOS: nopaline synthase promoter 0817/00006 12 also drawn in are those restriction cleavage sites which cut the vector only once.

Suitable promoters for the expression cassette are in principle all promoters able to control expression of foreign genes in plants. Preferably used is, in particular, a plant promoter or a promoter derived from a plant virus. The CaMV 35S promoter from cauliflower mosaic virus (Franck et al., Cell 21 (1980), 285-294) is particularly preferred. This promoter contains, as is known, different recognition sequences for transcriptional effectors which, in their totality, lead to permanent and constitutive expression of the inserted gene (Benfey et al., EMBO J. 8 (1989), 2195-2202).

The expression cassette may also contain a chemically inducible promoter by which expression of the exogenous DOXS or GGPPOR gene in the plant can be controlled at a particular time. Promoters of this type, such as the PRP1 promoter (Ward et al., Plant. Mol.

Biol. 22 (1993), 361-366), a promoter inducible by salicylic acid (WO 95/19443), a benzenesulfonamide-inducible (EP-A 388186), a tetracycline-inducible (Gatz et al., (1992) Plant J. 2, 397-404), an abscisic acid-inducible (EP-A 335528) and an ethanol- or cyclohexanone-inducible (WO 93/21334) promoter, inter alia, can be used.

Further particularly preferred promoters are those which ensure expression in tissues or parts of plants in which the biosynthesis of tocopherol or its precursors takes place. Particular mention should be made of promoters which ensure leaf-specific expression. Mention should be made of the promoter of cytosolic FBPase from potato or the ST-LSI promoter from potato (Stockhaus et al., EMBO J. 8 (1989) 2445 245).

An expression cassette is produced by fusing a suitable promoter to a suitable DOXS or GGPPOR DNA sequence and, preferably, to a DNA which is inserted between promoter and DOXS or GGPPOR DNA sequence and which codes for a chloroplast-specific transit peptide, and to a polyadenylation signal, by conventional recombination and cloning techniques as described, for example, in T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and in T.J. Silhavy, M.L. Berman and L.W.

Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and in Ausubel, F.M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience (1987).

0817/00006 13 It is also possible to use expression cassettes whose DNA sequence codes for a DOXS or GGPPOR fusion protein, where part of the fusion protein is a transit peptide which controls translocation of the polypeptide. Transit peptides specific for chloroplasts are particularly preferred, and these are eliminated enzymatically from the DOXS or GGPPOR part after translocation of the DOXS or GGPPOR gene into the chloroplasts. The particularly preferred transit peptide is derived from the plastidic transketolase (TK) or a functional equivalent of this transit peptide the transit peptide of the small subunit of rubisco or ferredoxin-NADP oxidoreductase).

The fused expression cassette coding for a DOXS gene or a GGPPOR gene is preferably cloned into a vector, for example pBinl9, which is suitable for transforming Agrobacterium tumefaciens.

The invention further relates to the use of an expression cassette comprising DNA sequences SEQ ID No. 1 or SEQ-ID No. 3 and SEQ ID No. 7 or DNA sequences hybridizing with the latter for the transformation of plants or cells, tissues or parts of plants. The preferred aim of the use is to increase the tocopherol, vitamin K, chlorophyll and carotenoid contents of the plant.

It is moreover possible, depending on the choice of the promoter, for expression to take place specifically in the leaves, in the seeds or other parts of the plant. The present invention further relates to such transgenic plants, propagation material thereof and cells, tissues or parts of these plants.

The invention additionally relates to transgenic plants transformed with an expression cassette comprising the sequence SEQ ID No. 1 or SEQ ID No. 3 and SEQ ID No. 7 or DNA sequences hybridizing with the latter, and transgenic cells, tissues, parts and propagation material of such plants. Particular preference is given in this connection to transgenic crop plants such as, for example, barley, wheat, rye, corn, oats, soybean, rice, cotton, sugarbeet, canola, sunflower, flax, hemp, potato, tobacco, tomato, oilseed rape, alfalfa, lettuce and the various tree, nut and vine species.

The invention further relates to: Process for transforming a plant, which comprises introducing expression cassettes comprising a DNA sequence SEQ ID No. 1 or a DNA sequence SEQ ID No. 3 and a SEQ ID No. 7 or DNA 0817/00006 14 sequences hybridizing with the latter into a plant cell, into callus tissue, a whole plant or protoplasts of plants, Use of the DNA sequence SEQ ID No. 1 or SEQ ID No. 3 and SEQ ID No. 7 or DNA sequences hybridizing with the latter to produce plants with increased tocopherol, vitamin K, chlorophyll and/or carotenoid contents by expression of a DOXS and a GGPPOR DNA sequence in plants.

The object have also been achieved by overexpression of a synthase (DOXS) gene, a p-hydroxyphenylpyruvate dioxygenase (HPPD) gene and a geranylgeranyl-pyrophosphate oxidoreductase (GGPPOR) gene in the plants, see Figure 1.

In order to increase the metabolite flux from primary metabolism into isoprenoid metabolism, the production of IPP as general starting substrate for all plastidic isoprenoids was increased.

For this purpose, the DOXS activity was increased by overexpression of the DOXS from E. coli in transgenic tobacco and oilseed rape plants. This can also be achieved by expressing homologous or other heterologous DOXS genes such as, for example, a DNA sequence SEQ ID No. 1.

The D-1-deoxy-xylulose 5-phosphate which is now available in increased quantities is converted further in the direction of geranylgeranyl pyrophosphate.

In order to convert the GGPP which is now available in increased quantities in the direction of tocopherols and carotenoids, in a further step essential to the invention in addition the activity of the enzyme geranylgeranyl pyrophosphate oxidoreductase is increased by overexpression of a corresponding homologous or heterologous gene. This measure achieves an increased production of phytyl pyrophosphate through increased conversion of geranylgeranyl pyrophosphate into phytyl pyrophosphate.

This done, for example, by enhanced expression of the GGPPOR gene from Arabidopsis thaliana (SEQ ID No. 7) in transgenic plants. In order to ensure plastidic localization, a transit signal sequence is put in front of the Arabidopsis GGPPOR. Also suitable as expression cassette is a DNA sequence coding for a GGPPOR gene which hybridizes with SEQ ID No. 7 and which is derived from other organisms or from other plants.

0817/00006 Example 15 describes the cloning of the GGPPOR gene from Arabidopsis thaliana.

In order to convert the PPP which is now available in increased quantities in the direction of tocopherol and carotenoids, in a further step essential to the invention in addition the activity of the enzyme p-hydroxylphenylpyruvate dioxygenase (HPPD) is increased by overexpression of a corresponding homologous or heterologous gene. This measure achieves increased production of homogentisic acid by increased conversion of hydroxyphenylpyruvate into homogentisic acid.

cDNAs coding for this enzyme have been described from various organisms such as, for example, from microorganisms, from plants and from humans.

Example 10 describes the cloning of the HPPD gene from Streptomyces avermitilis (Denoya et al., J. Bacteriol. 176(1994), 5312-5319; SEQ ID No. In order to ensure a plastidic localization, a transit signal sequence is put in front of the Streptomyces HPPD. Also suitable as expression cassette is a DNA sequence which codes for an HPPD gene which hybridizes with SEQ ID No. 5 and is derived from other organisms or from plants.

The increase in the plastidic D-l-deoxy-xylulose 5-phosphate, the phytyl pyrophosphate and the homogentisic acid production leads to increased production of all plastidic isoprenoids. The increased provision of these precursors ensures that sufficient substrate is available for the production of tocopherols, chlorophylls, vitamin K and phylloquinones in the plastids.

The transgenic plants according to the invention are produced by transforming the plants with a construct containing the DOXS, the HPPD gene and the GGPPOR gene (Example 17). Tobacco and oilseed rape were employed as model plants for producing tocopherols, vitamin K, chlorophylls and carotenoids.

The invention relates to the use of the DNA sequences SEQ ID No.

1 or SEQ ID No. 3, SEQ ID No. 5 and SEQ-ID No. 7, which code for a DOXS, an HPPD and a GGPPOR or functional equivalents thereof to produce a plant with increased tocopherol, vitamin K, chlorophyll and/or carotenoid contents. The nucleic acid sequences may in these cases be, for example, DNA or cDNA sequences. Coding sequences suitable for insertion into an expression cassette are, for example, those coding for a DOXS, an HPPD and a GGPPOR and conferring on the host the ability to overproduce tocopherol.

0817/00006 16 The expression cassettes additionally comprise regulatory nucleic acid sequences which control the expression of the coding sequence in the host cell. In a preferred embodiment, an expression cassette comprises upstream, i.e. at the 5' end of the coding sequence, a promoter and downstream, i.e. at the 3' end, a polyadenylation signal and, where appropriate, further regulatory elements which are operatively linked to the coding sequence for the DOXS, the HPPD or the GGPPOR gene located in between.

Operative linkage means sequential arrangement of promoter, coding sequence, terminator and, where appropriate, further regulatory elements in such a way that each of the regulatory elements can properly carry out its function in the expression of the coding sequence. The sequences which are preferred for the operative linkage, but are not restricted thereto, are targeting sequences to ensure subcellular localization in the apoplast, in the vacuole, in plastids, in the mitochondrion, in the endoplasmic reticulum in the cell nucleus, in elaioplasts or other compartments and translation enhancers such as the leader sequence from tobacco mosaic virus (Gallie et al., Nucl.

Acids Res. 15 (1987), 8693-8711).

For example, the plant expression cassette can be incorporated into the tobacco transformation vector pBinAR-Hyg. Fig. 2 shows the tobacco transformation vectors pBinAR-Hyg with the promoter and pBinAR-Hyg with the seed-specific promoter phaseolin 796 HPT: hygromycin phosphotransferase OCS: octopine synthase terminator PNOS: nopaline synthase promoter also drawn in are those restriction cleavage sites which cut the vector only once.

Suitable promoters for the expression cassette are in principle all promoters able to control expression of foreign genes in plants. Preferably used is, in particular, a plant promoter or a promoter derived from a plant virus. The CaMV 35S promoter from cauliflower mosaic virus (Franck et al., Cell 21 (1980), 285-294) is particularly preferred. This promoter contains, as is known, different recognition sequences for transcriptional effectors which, in their totality, lead to permanent and constitutive expression of the inserted gene (Benfey et al., EMBO J. 8 (1989), 2195-2202).

The expression cassette may also contain a chemically inducible promoter by which expression of the exogenous DOXS, HPPD and nGGPOR gene in the plant can be controlled at a particular time.

0817/00006 17 Promoters of this type, such as the PRP1 promoter (Ward et al., Plant. Mol. Biol. 22 (1993), 361-366), a promoter inducible by salicylic acid (WO 95/19443), a benzenesulfonamide-inducible (EP-A 388186), a tetracycline-inducible (Gatz et al., (1992) Plant J. 2, 397-404), an abscisic acid-inducible (EP-A 335528) and an ethanol- or cyclohexanone-inducible (WO 93/21334) promoter, inter alia, can be used.

Further particularly preferred promoters are those which ensure expression in tissues or parts of plants in which the biosynthesis of tocopherol or its precursors takes place. Particular mention should be made of promoters which ensure leaf-specific expression. Mention should be made of the promoter of cytosolic FBPase from potato or the ST-LSI promoter from potato (Stockhaus et al., EMBO J. 8 (1989) 2445 245).

An expression cassette is produced by fusing a suitable promoter to a suitable DOXS, HPPD and GGPPOR DNA sequence and, preferably, to a DNA which is inserted between promoter and DOXS, HPPD and GGPOR DNA sequence and which codes for a chloroplast-specific transit peptide, and to a polyadenylation signal, by conventional recombination and cloning techniques as described, for example, in T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and in T.J. Silhavy, M.L. Berman and L.W.

Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and in Ausubel, F.M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience (1987).

It is also possible to use expression cassettes whose DNA sequence codes for a DOXS, HPPD and GGPOR fusion protein, where part of the fusion protein is a transit peptide which controls translocation of the polypeptide. Transit peptides specific for chloroplasts are particularly preferred, and these are eliminated enzymatically from the DOXS, HPPD and GGPPOR part after translocation of the DOXS, HPPD and GGPOR gene into the chloroplasts. The particularly preferred transit peptide is derived from the plastidic transketolase (TK) or a functional equivalent of this transit peptide the transit peptide of the small subunit of rubisco or ferredoxin-NADP oxidoreductase).

The fused expression cassette coding for a DOXS gene, an HPPD gene or a GGPPOR gene is preferably cloned into a vector, for example pBinl9, which is suitable for transforming Agrobacterium tumefaciens.

0817/00006 18 The invention further relates to the use of an expression cassette comprising DNA sequences SEQ ID No. 1 or SEQ-ID No. 3, SEQ ID No. 5 and SEQ ID No. 7 or DNA sequences hybridizing with the latter for the transformation of plants or cells, tissues or parts of plants. The preferred aim of the use is to increase the tocopherol, vitamin K, chlorophyll and carotenoid contents of the plant.

It is moreover possible, depending on the choice of the promoter, for expression to take place specifically in the leaves, in the seeds or other parts of the plant. The present invention further relates to such transgenic plants, propagation material thereof and cells, tissues or parts of these plants.

The invention additionally relates to transgenic plants transformed with an expression cassette comprising the sequence SEQ ID No. 1 or SEQ ID No. 3, SEQ ID No. 5 and SEQ ID No. 7 or DNA sequences hybridizing with the latter, and transgenic cells, tissues, parts and propagation material of such plants.

Particular preference is given in this connection to transgenic crop plants such as, for example, barley, wheat, rye, corn, oats, soybean, rice, cotton, sugarbeet, canola, sunflower, flax, hemp, potato, tobacco, tomato, oilseed rape, alfalfa, lettuce and the various tree, nut and vine species.

The invention further relates to: Processes for transforming a plant, which comprises introducing expression cassettes comprising a DNA sequence SEQ ID No. 1 or SEQ ID No. 3, a DNA sequence SEQ ID No. 5 and a DNA sequence SEQ ID No. 7 or DNA sequences hybridizing with the latter into a plant cell, into callus tissue, a whole plant or protoplasts of plants, Use of the DNA sequence SEQ ID No. 1 or SEQ ID No. 3, SEQ ID No. 5 and SEQ ID No. 7 or DNA sequences hybridizing with the latter to produce plants with increased tocopherol, vitamin K, chlorophyll and/or carotenoid contents by expression of a DOXS, an HPPD and an GGPPOR DNA sequence in plants.

It was therefore an additional object of the present invention to develop a test system for identifying DOXS inhibitors.

0817/00006 19 This object has been achieved by expressing a DOXS gene from Arabidopsis or E. coli, or DNA sequences hybridizing therewith, and subsequently testing chemicals for inhibition of the DOXS enzyme activity.

The transgenic plants are produced by transforming the plants with a construct containing the DOXS gene. Arabidopsis and oilseed rape were employed as model plants for the production of tocopherols, vitamin K, chlorophylls and carotenoids.

Cloning of the complete DOXS gene from Arabidopsis takes place by isolating the cDNA (SEQ ID No. 1) specific for the DOXS gene.

The invention relates to the use of the DNA sequence SEQ ID No. 1 or SEQ ID No. 3 which codes for a DOXS or functional equivalent thereof for producing a plant with increased tocopherol, vitamin K, chlorophyll and/or carotenoid content. The nucleic acid sequence can moreover be, for example, a DNA or cDNA sequence.

Examples of coding sequences suitable for insertion into an expression cassette are those which code for a DOXS and which confer on the host the ability to overproduce tocopherol.

The expression cassettes additionally comprise regulatory nucleic acid sequences which control the expression of the coding sequence in the host cell. In a preferred embodiment, an expression cassette comprises a promoter upstream, i.e. at the end of the coding sequence, and a polyadenylation signal downstream, i.e. at the 3' end, and, where appropriate, further regulatory elements which are operatively linked to the coding sequence for the DOXS gene located in between. Operative linkage means sequential arrangement of promoter, coding sequence, terminator and, where appropriate, further regulatory elements in such a way that each of the regulatory elements can properly carry out its function in the expression of the coding sequence.

The sequences which are preferred for the operative linkage, but are not restricted thereto, are targeting sequences to ensure subcellular localization in the apoplast, in the vacuole, in plastids, in the mitochondrion, in the endoplasmic reticulum in the cell nucleus, in elaioplasts or other compartments and translation enhancers such as the 5' leader sequence from tobacco mosaic virus (Gallie et al., Nucl. Acids Res. 15 (1987) 8693 8711).

For example, the plant expression cassette can be incorporated into the tobacco transformation vector pBinAR-Hyg. Fig. [lacuna] shows the tobacco transformation vectors pBinAR-Hyg with the 0817/00006 promoter and pBinAR-Hyg with the seed-specific promoter phaseolin 796 HPT: hygromycin phosphotransferase OCS: octopine synthase terminator PNOS: nopaline synthase promoter also drawn in are those restriction cleavage sites which cut the vector only once.

Suitable promoters for the expression cassette are in principle all promoters able to control expression of foreign genes in plants. Preferably used is, in particular, a plant promoter or a promoter derived from a plant virus. The CaMV 35S promoter from cauliflower mosaic virus (Franck et al., Cell 21 (1980), 285-294) is particularly preferred. This promoter contains, as is known, different recognition sequences for transcriptional effectors which, in their totality, lead to permanent and constitutive expression of the inserted gene (Benfey et al., EMBO J. 8 (1989), 2195-2202).

The expression cassette may also contain a chemically inducible promoter by which expression of the exogenous DOXS gene in the plant can be controlled at a particular time. Promoters of this type, such as the PRP1 promoter (Ward et al., Plant. Mol. Biol.

22 (1993), 361-366), a promoter inducible by salicylic acid (WO 95/19443), a benzenesulfonamide-inducible (EP-A 388186), a tetracycline-inducible (Gatz et al., (1992) Plant J. 2, 397-404), an abscisic acid-inducible (EP-A 335528) and an ethanol- or cyclohexanone-inducible (WO 93/21334) promoter, inter alia, can be used.

Further particularly preferred promoters are those which ensure expression in tissues or parts of plants in which the biosynthesis of tocopherol or its precursors takes place. Particular mention should be made of promoters which ensure leaf-specific expression. Mention should be made of the promoter of cytosolic FBPase from potato or the ST-LSI promoter from potato (Stockhaus et al., EMBO J. 8 (1989) 2445 245).

It has been possible with the aid of a seed-specific promoter to express a foreign protein stably up to a content of 0.67% of the total soluble seed protein in the seeds of transgenic tobacco plants (Fiedler and Conrad, Bio/Technology 10 (1995), 1090-1094).

The expression cassette can therefore contain, for example, a seed-specific promoter (preferably the phaseolin promoter (US 5504200), the USP (Baumlein, H. et al. Mol. Gen. Genet.

(1991) 225 459 467) or LEB4 promoter (Fiedler and Conrad, 0817/00006 21 1995)), the LEB4 signal peptide, the gene to be expressed, and an ER retention signal. The construction of a cassette of this type is depicted diagrammatically by way of example in Figure 2.

An expression cassette is produced by fusing a suitable promoter to a suitable DOXS DNA sequence and, preferably, to a DNA which is inserted between promoter and DOXS DNA sequence and which codes for a chloroplast-specific transit peptide, and to a polyadenylation signal, by conventional recombination and cloning techniques as described, for example, in T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and in T.J. Silhavy, M.L. Berman and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and in Ausubel, F.M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc.

and Wiley-Interscience (1987).

Particularly preferred sequences are those which ensure targeting in the apoplast, in plastids, in the vacuole, in the mitochondrion, in the endoplasmic reticulum (ER) or, due to absence of appropriate operative sequences, ensure retention in the compartment of production, the cytosol (Kermode, Crit. Rev.

Plant Sci. 15, 4 (1996), 285 423). Localization in the ER has proved particularly beneficial for the amount of protein accumulated in transgenic plants (Schouten et al., Plant Mol.

Biol. 30 (1996), 781 792).

It is also possible to use expression cassettes whose DNA sequence codes for a DOXS fusion protein, where part of the fusion protein is a transit peptide which controls translocation of the polypeptide. Transit peptides specific for chloroplasts are particularly preferred, and these are eliminated enzymatically from the DOXS part after translocation of the DOXS gene into the chloroplasts. The particularly preferred transit peptide is derived from the plastidic transketolase (TK) or a functional equivalent of this transit peptide the transit peptide of the small subunit of rubisco or ferredoxin-NADP oxidoreductase).

The inserted nucleotide sequence coding for a DOXS can be prepared by synthesis or be obtained naturally or comprise a mixture of synthetic and natural DNA constituents, and may consist of different heterologous DOXS gene sections from different organisms. In general, synthetic nucleotide sequences are produced with codons preferred by plants. These codons preferred by plants can be identified from codons with the highest protein frequency which are expressed in most plant 0817/00006 22 species of interest. For preparing an expression cassette it is possible to manipulate various DNA fragments in order to obtain a nucleotide sequence which expediently reads in the correct direction and is equipped with a correct reading frame. Adapters or linkers can be attached to the fragments for connecting the DNA fragments to one another.

It is possible and expedient for the promoter and terminator regions to be provided in the direction of transcription with a linker or polylinker which contains one or more restriction sites for inserting this sequence. As a rule, the linker has 1 to usually 1 to 8, preferably 2 to 6, restriction sites. The linker generally has a size of less than 100 bp, frequently less than bp, but at least 5 bp, inside the regulatory regions. The promoter may be both native or homologous and foreign or heterologous to the host plant. The expression cassette comprises in the direction of transcription the promoter, a DNA sequence which codes for a DOXS gene, and a region for termination of transcription. Various termination regions are interchangeable as desired.

It is furthermore possible to employ manipulations which provide appropriate restriction cleavage sites or delete the redundant DNA or restriction cleavage sites. It is possible in relation to insertions, deletions or substitutions, e.g. transitions and transversions, to use in vitro mutagenesis, primer repair, restriction or ligation. It is possible with suitable manipulations, e.g. restriction, chewing back or filling in overhangs for blunt ends, to provide complementary ends of the fragments for ligation.

It may be important for success according to the invention inter alia to attach the specific ER retention signal SEKDEL (Schouten, A. et al. Plant Mol. Biol. 30 (1996), 781 792), whereby the average level of expression is tripled or quadrupled. It is also possible to employ other retention signals which naturally occur with plant and animal proteins which are localized within the ER for constructing the cassette.

Preferred polyadenylation signals are plant polyadenylation signals, preferably those which essentially correspond to T-DNA polyadenylation signals from Agrobacterium tumefaciens, especially of gene 3 of the T-DNA (octopine synthase) of the Ti plasmid pTiACH5 (Gielen et al., EMBO J. 3 (1984) 835 ff) or functional equivalents.

0817/00006 23 An expression cassette may comprise, for example, a constitutive promoter (preferably the CaMV 35 S promoter), the LeB4 signal peptide, the gene to be expressed, and the ER retention signal.

The ER retention signal preferably used is the amino acid sequence KDEL (lysine, aspartic acid, glutamic acid, leucine).

The fused expression cassette which codes for a DOXS gene is preferably cloned into a vector, for example pBinl9, which is suitable for transforming Agrobacterium tumefaciens. Agrobacteria transformed with such a vector can then be used in a known manner for transforming plants, in particular crop plants, e.g. tobacco plants, by, for example, bathing wounded leaves or pieces of leaf in a solution of agrobacteria and then cultivating in suitable media. The transformation of plants by agrobacteria is dislosed inter alia by F.F. White, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1, Engineering and Utilization, edited by S.D. Kung and R. Wu, Academic Press, 1993, pp. 15-38. Transgenic plants containing a gene, integrated in the expression cassette, for expression of a DOXS gene can be regenerated in a known manner from the transformed cells of the wounded leaves or pieces of leaf.

For transformation of a host plant with a DNA coding for a DOXS, an expression cassette is incorporated as insertion into a recombinant vector whose vector DNA comprises additional functional regulatory signals, for example sequences for replication or integration. Suitable vectors are described inter alia in "Methods in Plant Molecular Biology and Biotechnology" (CRC Press), Chap. 6/7, pp. 71-119 (1993).

It is possible by using the recombination and cloning techniques cited above to clone the expression cassettes into suitable vectors which make their replication possible, for example in E.

coli. Suitable cloning vectors are, inter alia, pBR332, pUC series, M13mp series and pACYC184. Binary vectors able to replicate both in E. coli and in agrobacteria are particularly suitable.

The invention further relates to the use of an expression cassette comprising a DNA sequence SEQ No. 1 or SEQ ID No. 3; SEQ ID No. 1 or SEQ ID No. 3 and SEQ ID No. 5; SEQ ID No. 1 or SEQ-ID No. 3 and SEQ-ID No. 7 or a DNA sequence SEQ ID No. 1 or SEQ ID No. 3 and SEQ ID No. 5 and SEQ ID No. 7, or DNA sequences hybridizing with the latter for transforming plants, or cells, tissues or parts of plants. The aim of the use is preferably to 0817/00006 24 increase the tocopherol, vitamin K, chlorophyll and carotenoid contents of the plant.

It is moreover possible, depending on the choice of the promoter, for expression to take place specifically in the leaves, in the seeds, or other parts of the plant. The present invention further relates to such transgenic plants, to propagation material thereof and to cells, tissues or parts of the plants.

The expression cassette can in addition be employed for transforming bacteria, cyanobacteria, yeasts, filamentous fungi and algae with the aim of increasing tocopherol, vitamin K, chlorophyll and/or carotenoid production.

The transfer of foreign genes into the genome of a plant is referred to as transformation. In this connection, the described methods for transforming and regenerating plants from plant tissues or plant cells are utilized for transient or stable transformation. Suitable methods are protoplast transformation by polyethylene glycol-induced DNA uptake, the biolistic method using the gene gun called the particle bombardment method, electroporation, incubation of dry embryos in DNA-containing solution, microinjection and gene transfer mediated by Agrobacterium. Said processes are described, for example, in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, edited by S.D. Kung and R. Wu, Academic Press (1993) 128-143 and in Potrykus Annu.

Rev. Plant Physiol. Plant Molec. Biol. 42 (1991) 205-225). The construct to be expressed is preferably cloned into a vector which is suitable for transforming Agrobacterium tumefaciens, for example pBinl9 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711).

Agrobacteria transformed with an expression cassette can likewise be used in a known manner for transforming plants, in particular crop plants such as cereals, corn, oats, soybean, rice, cotton, sugarbeet, canola, sunflower, flax, hemp, potato, tobacco, tomato, oilseed rape, alfalfa, lettuce and the various tree, nut and vine species, e.g. by bathing wounded leaves or pieces of leaf in a solution of agrobacteria and subsequently cultivating in suitable media.

Functionally equivalent sequences which code for a DOXS gene are sequences which, despite differing in nucleotide sequence, still have the required functions. Functional equivalents thus comprise naturally occurring variants of the sequences described herein, and artificial artificial nucleotide sequences obtained, for 0817/00006 example, by chemical synthesis and adapted to the codon usage of a plant.

A functional equivalent also means in particular natural or artificial mutations of an originally isolated sequence coding for a DOXS, which still show the required funtion. Mutations comprise substitutions, additions, deletions, transpositions or insertions of one or more nucleotide residues. Thus, the present invention also includes, for example, nucleotide sequences which are obtained by modifying the DOXS nucleotide sequence. The aim of such a modification may be, for example, to localize further the coding sequence present therein or else, for example, to insert further restriction enzyme cleavage sites.

Functional equivalents are also variants whose function is attenuated or enhanced by comparison with the initial gene or gene fragment.

Artificial DNA sequences are also suitable as long as they confer, as described above, the required property, for example of increasing the tocopherol content in the plant by overexpression of the DOXS gene in crop plants. Such artificial DNA sequences can be identified, for example, by back-translation of proteins which have been constructed by molecular modelling and have DOXS activity, or by in vitro selection. Particularly suitable coding DNA sequences are those which have been obtained by backtranslation of a polypeptide sequence in accordance with the codon usage specific for the host plant. The specific codon usage can easily be established by a skilled worker familiar with plant genetic methods through computer analyses of other, known genes in the plant to be transformed.

Further suitable equivalent nucleic acid sequences which should be mentioned are sequences which code for fusion proteins, in which case a plant DOXS polypeptide or a functionally equivalent part thereof is a constituent of the fusion protein. The second part of the fusion protein can be, for example, another polypeptide with enzymatic activity, or an antigenic polypeptide sequence with whose aid it is possible to detect DOXS expression myc tag or his tag). However, this is preferably a regulatory protein sequence, e.g. a signal or transit peptide which guides the DOXS protein to the required site of action.

However, the invention also relates to the expression products generated according to the invention, and to fusion proteins composed of a transit peptide and a polypeptide with DOXS 0817/00006 26 activity.

Increasing the tocopherol, vitamin K, chlorophyll and/or carotenoid content means for the purpose of the present invention the artificially acquired capability of increased activity in the biosynthesis of these compounds through functional overexpression of the DOXS gene in the plant compared with the plant which has not been genetically modified, for the duration of at least one plant generation.

The site of tocopherol biosynthesis is generally the leaf tissue so that leaf-specific expression of the DOXS gene is sensible.

However, it is obvious that tocopherol biosynthesis need not be confined to the leaf tissue, but may also take place tissue-specifically in all other parts of the plant for example in oilbearing seeds.

Constitutive expression of the exogenous DOXS gene is an additional advantage. However, on the other hand, inducible expression may also appear to be desirable.

The effectiveness of expression of the transgenically expressed DOXS gene can be determined, for example, in vitro by shoot meristem propagation. In addition, an alteration in the nature and level of the expression of the DOXS gene and its effect on tocopherol biosynthesis activity can be tested in glasshouse experiments on test plants.

The invention additionally relates to transgenic plants transformed with an expression cassette comprising the sequence SEQ ID No.l or SEQ ID No. 3; SEQ ID No. 1 or SEQ ID No. 3 and SEQ No. 5; SEQ ID No. 1 or SEQ ID No. 3 and SEQ ID No. 7 or a DNA sequence SEQ ID No. 1 or SEQ ID No. 3 and SEQ ID No. 5 and SEQ ID No. 7, or DNA sequences hybridizing with the latter, and transgenic cells, tissues, parts and propagation material of such plants. Particularly preferred in this connection are transgenic crop plants such as, for example, barley, wheat, rye, corn, oats, soybean, rice, cotton, sugarbeet, canola, sunflower, flax, hemp, potato, tobacco, tomato, oilseed rape, alfalfa, lettuce and the various tree, nut and vine species.

Plants for the purpose of the invention are mono- and dicotyledonous plants or algae.

In order to be able to find efficient DOXS inhibitors, it is necessary to provide suitable test systems with which inhibitor/ enzyme binding studies can be carried out. For this purpose, for 0817/00006 27 example, the complete cDNA sequence of DOXS from Arabidopsis is cloned into an expression vector (pQE, Qiagen) and overexpressed in E. coli.

The DOXS protein expressed using the expression cassette is particularly suitable for finding inhibitors specific for DOXS.

For this purpose, DOXS can be employed, for example, in an enzyme assay in which the activity of DOXS is determined in the presence and absence of the active substance to be tested. Comparison of the two activity determinations allows qualitative and quantitative information to be obtained about the inhibitory behavior of the active substance to be tested. Methods for DOXS activity determination are described (Putra et. al., Tetrahedron Letters 39 (1998), 23-26; Sprenger et al., PNAS 94 (1997), 12857-12862).

The test system according to the invention can be used to examine rapidly and simply a large number of chemical compounds for inhibitory properties. The method allows reproducible selection, from a large number of substances, specifically of those with high activity in order subsequently to carry out with these substances further, more intensive tests familiar to the skilled worker.

It is possible in principle by overexpression of the gene sequence SEQ ID NO: 1 or SEQ ID NO: 3 coding for a DOXS in a plant to achieve increased resistance to DOXS inhibitors. The invention likewise relates to transgenic plants produced in this way.

The invention further relates to: A process for transforming a plant, which comprises introducing an expression cassette comprising a DNA sequence SEQ ID No. 1 or SEQ ID No. 3 or a DNA sequence hybridizing with the latter into a plant cell, into callus tissue, a whole plant or protoplasts of plants.

The use of a plant for producing plant DOXS.

The use of the expression cassette comprising a DNA sequence SEQ ID No. 1 or SEQ ID NO. 3 or a DNA sequence hybridizing with the latter for producing plants with increased resistance to DOXS inhibitors by enhanced expression of a DNA 0817/00006 28 sequence SEQ ID No. 1 or SEQ ID N'o 3 or a DNA sequence hybridizing with the latter.

The use of the DNA sequence SEQ ID No. 1 or SEQ ID NO. 3 or of a DNA sequence hybridizing with the latter for producing plants with increased tocopherol, vitamin K, chlorophyll and/or carotenoid content by expression of a DOXS DNA sequence in plants.

The use of the expression cassette comprising a DNA sequence SEQ ID No. 1 or SEQ ID NO: 3 or a DNA sequence hybridizing with the latter for producing a test system for identifying DOXS inhibitors.

The invention is illustrated by the examples which now follow, but is not confined to these: General cloning methods The cloning steps carried out for the purpose of the present invention, such as restriction cleavages, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linkage of DNA fragments, transformation of E. coli cells, cultivation of bacteria, replication of phages and recombinant DNA sequence analysis were carried out as described in Sambrook et al. (1989) Cold Spring Harbor Laboratory Press; ISBN 0-87969-309-6).

The bacterial strains coli, XL-I Blue) used below were purchased from Stratagene. The agrobacterium strain used for plant transformation (Agrobacterium tumefaciens, C58C1 with the plasmid pGV2260 or pGV3850kann) has been described by Deblaere et al. in (Nucl. Acids Res. 13 (1985) 4777). Alternative possibilities are also to employ the agrobacterium strain LBA4404 (Clontech) or other suitable strains. Vectors which can be used for cloning are pUC19 (Yanish-Perron, Gene 33 (1985), 103-119) pBluescript SK- (Stratagene), pGEM-T (Promega), pZerO (Invitrogen), pBinl9 (Bevan et al., Nucl. Acids Res. 12 (1984), 8711-8720) and pBinAR (H6fgen and Willmitzer, Plant Science 66 (1990), 221-230).

Recombinant DNA sequence analysis Recombinant DNA molecules were sequenced using a Licor laser fluorescence DNA sequencer (marketed by MWG Biotech, Ebersbach) 0817/00006 29 using the Sanger method (Sanger et al., Proc. Natl. Acad. Sci.

USA 74 (1977), 5463-5467).

Example 1 Production of the Arabidopsis thaliana DOXS transformation constructs The Arabidopsis thaliana DOXS gene was cloned as described in Mandel et al. (1996) as complete cDNA into the vector pBluescript KS- (Stratagene).

To produce overexpression constructs, a 2.3 kb fragment (designated F-23-C) was isolated via the pBluescript KS- Hincll (blunt-end) and Sad cleavage sites. This sequence contains the complete DOXS cDNA from the ATG start codon to the EcoRlI cleavage site located 80 bp downstream of the stop codon. This fragment was cloned via the Smal (blunt-end) and Sad cleavage sites into the pBIN19 vector (Figure 3) (Bevan et al., (1980) which contains the 35S promoter of cauliflower mosaic virus (Franck et al., Cell 21(1), 285-294 (1980)) arranged three times in sequence.

To produce antisense constructs, a region of the 3' end of the cDNA (called F-23-C antisense) was cloned into the abovementioned pBIN19-3X35S vector. Part of the 5' region of the DOXS cDNA in pBluescript KS- was digested via Hincll and the DOXS-internal BglII cleavage site, and the resulting fragment was removed.

(Figure The BglII cleavage site was filled in by the Klenow fill-in reaction (Klenow polymerase; Roche; after reaction according to manufacturer's protocol) so that a blunt end was produced. The ends which were now compatible (BglII blunt end and HinclII were ligated. The 3' region of the DOXS cDNA was then cloned via KpnI and Xbal (both cleavage sites are located in the polylinker of pBluescript KS-5' and 3' of the DOXS cDNA) in antisense orientation into the pBIN19 vector described above in antisense orientation.

Transformations of Arabidopsis thaliana plants with the constructs described above using Agrobacterium tumefaciens took place by the vacuum infiltration method (Bent et al., Science 265 (1994), 1856-1860). Several independent transformands were isolated for each construct. Each letter (see Table 1) denotes an independent transformed line. Plants from the Tl generation obtained therefrom were examined for homo- or heterozygosity.

Several plants from each line were crossed in order to carry out Sa segregation analysis. The number in Table 1 corresponds to the 0817/00006 individual plant selected for further analyses. Both homo- and heterozygous lines were obtained. The segregation analysis of the resulting lines is shown in Table 1 below: Table 1. Segregation analysis of the transgenic DOXS T2 plants LINES SEGREGATION A9 A19 100% Bll B4 100% C2 100% D3 D17 100% E9 E14 100% F9 2 F14 100% Example 2 Isolation of genomic DNA of the bacterium Escherichia coli XL1 2Blue A culture of Escherichia coli XL1 Blue was grown in 300 ml of Luria broth medium at 37 0 C for 12 hours. The genomic DNA of the bacterium was isolated from this culture by first pelleting it at 5 000 revolutions in a Sorvall RC50 fuge. The pellet was then resuspended in 1/30 of the volume of the original culture of lysis buffer (25 mM EDTA, 0.5% SDS; 50 mM Tris HC1, pH An equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) was added and incubated at 70 degrees for 10 minutes. The aqueous phase was then separated from the phenolic in a Heraeus floor centrifuge at 3 500 rev for 15 minutes. The aqueous supernatant was mixed with 2.5 volumes of ethanol and 1/10 volume of 8 M lithium chloride, and the nucleic acids were precipitated at room temperature for 10 minutes. The pellet was then taken up in 400 il of TE/RNAse and incubated at 37 degrees for 10 minutes.

The solution was again shaken with one volume of phenol/ chloroform/isoamyl alcohol (25:24:1), and the supernatant was precipitated with 2.5 volumes of ethanol and 1/10 volume of 8 M lithium chloride. The pellet was then washed with 80% ethanol and taken up in 400 p1 of TE/RNAse.

0817/00006 31 Example 3 Isolation of the DOXS from E. coli Oligonucleotides for a PCR were derived from the DOXS DNA sequence (Acc. Number AF035440), and a BamHI restriction cleavage site was attached to them at the 5' end, and an XbaI or another BamHI restriction cleavage site was attached to them at the 3' end. The oligonucleotide at the 5' end comprises the sequence 5'-ATGGATCCATGAGTTTT-GATATTGCCAAATAC-3' (nucleotides 1-24 of the DNA sequence; in italics) starting with the ATG start codon of the gene, and the oligonucleotide at the 3' end comprises the sequence 5'-ATTCTAGATTATGCCAGCCAGGCCTTG-3' or 5'-ATGGATCCTTATGCCAGCCAGGCCTTG-3' (nucleotides 1845-1863 of the reverse complementary DNA sequence; in italics) starting with the stop codon of the gene. The PCR reaction with the two BamHI-containing oligonucleotides was carried out with Pfu polymerase (Stratagene GmbH, Heidelberg) in accordance with the manufacturer's information. 500 ng of the genomic DNA from E.

coli were employed as template. The PCR program was as follows: cycles: 4 sec 94°C, 30 sec 52 0 C, 2 min 72 0

C;

cycles: 4 sec 94°C, 30 sec 48 0 C, 2 min 72 0

C;

cycles: 4 sec 94 0 C, 30 sec 44 0 C, 2 min 72°C The fragment was purified using a Gene-Clean kit (Dianova GmbH, Hilden) and cloned in accordance with the manufacturer's information into the vector PCR-Script (Stratagene GmbH, Heidelberg). The correctness of the sequence was established by sequencing. The fragment was BamHI isolated from the PCR-Script vector and ligated into a correspondingly cut Binl9 vector which additionally contains the transit peptide of potato transketalase downstream of the CaMV 35S as promoter. The transit peptide ensures plastidic localization. The constructs are depicted in Figure 5 and 6, and the fragments have the following significance: Fragment A (529 bp) comprises the 35S promoter of cauliflower mosaic virus (nucleotides 6909 to 7437 of cauliflower mosaic virus). Fragment B (259 bp) comprises the transit peptide of transketolase. Fragment E comprises the DOXS gene. Fragment D (192 bp) comprises the polyadenylation signal of gene 3 of the T DNA of the Ti plasmid pTIACH5 (Gielen et al., 1984) to terminate transcription.

0817/00006 32 The PCR reaction with the 5'-BamHI and 3'-XbaI-containing oligonucleotides was carried out with Taq polymerase (Takara, Sosei Co., Ltd.) in accordance with the manufacturer's information. 500 ng of the genomic DNA from E. coli were employed as template. The PCR program was as follows: cycles: 4 sec 94°C, 4 sec 50°C, 2 min cycles: 4 sec 94°C, 30 sec 46 0 C, 2 min 68 0

C

cycles: 4 sec 94C, 30 sec 42 0 C, 2 min 68 0

C

The fragment was purified using a Gene-Clean kit and ligated into the vector pGemT (Promega GmbH, Mannheim). It was cloned as BamHI/XbaI fragment into a correspondingly cut pBinl9AR vector downstream of the CaMV 35S promoter. The sequence was checked by sequencing (SEQ-ID No. This revealed two non-conservative base exchanges which, compared with the published sequence, lead to a change in amino acid 152 (asparagine) to valine and amino acid 330 (cysteine) to tryptophan.

Example 4 Detection of increased amounts of DOXS RNA in transgenic plants Total RNA from 15-day old seedlings of various transgenic lines possessing the DOXS overexpression construct was extracted by the method of Logeman et al., Anal. Biochem. 163, 16-20 (1987), fractionated in a 1.2% agarose gel, transferred to filters and hybridized with a 2.1 kb long DOXS fragment as probe (Figure 7).

Example Detection of increased amounts of DOXS protein in transgenic plants Total protein (Figure 8) from 15-day old seedlings of various independent transgenic plants possessing the DOXS overexpression construct was isolated and detected in a Western analysis using a polyclonal anti-DOXS antibody (IgG) (Figure 9).

Example 6 Measurement of the carotenoid and chlorophyll contents The total amounts of carotenoids and chlorophylls were determined as described by Lichtenthaler and Wellburn (1983) using 100% acetone extracts. The results of the multiple measurements of the 0817/00006 33 transgenic lines possessing the DOXS overexpression construct are shown in Table 2 below.

Table 2: Total carotenoid and chlorophyll contents of transgenic DOXS lines LINE TOTAL CHLOROPHYLLS TOTAL CAROTENOIDS clal mutant 5 Wild type 100 100 B-4 86 89 B-ll 84 C-2 98 107 D-3 128 135 D-17 136 149 E-14 121 139 F-7 80 F-14 85 107 Example 7 Transformation of oilseed rape The production of transgenic oilseed rape plants is based on a protocol of Bade, JB and Damm, B (in Gene, Transfer to Plants, Potrykus, I. and Spangenberg, eds, Springer Lab Manual, Springer Verlag, 1995, 30-38), in which the composition of the media used are also stated. The transformations took place with the agrobacterium strain LBA4404 (Clontech). The binary vectors used were the pBIN19 constructs with the complete DOXS cDNA already described above. The NOS terminator sequence in these pBIN vectors was replaced by the OCR terminator sequence.

Brassica napus seeds were surface-sterilized with 70% (v/v) ethanol, washed in H 2 0 at 550C for 10 min, incubated in 1% strength hypochlorite solution (25% v/v Teepol, 0.1% v/v Twenn for 20 min and washed six times with sterile H 2 0 for 20 min each time. The seeds were dried on filter paper for three days and 10-15 seeds were induced to germinate in a glass flask with 15 ml of germination medium. The roots and apices were removed from several seedlings (about 10 cm in size), and the remaining hypocotyls were cut into pieces about 6 mm long. The approx. 600 explants obtained in this way are washed with 50 ml of basal medium for 30 min and transferred into a 300 ml flask. After addition of 100 ml of callus induction medium, the cultures were incubated at 100 rpm for 24 h.

0817/00006 34 An overnight culture of the agrobacterium strain was set up in LB with kanamycin (20 mg/1) at 290C, and 2 ml of this were incubated in 50 ml of LB without kanamycin at 29 0 C for 4 h until the OD 600 was 0.4-0.5. After pelleting of the culture at 2 000 rpm for 25 min, the cell pellet was resuspended in 25 ml of basal medium.

The concentration of the bacteria in the solution was adjusted to an OD600 of 0.3 by adding further basal medium.

The callus induction medium was removed from the oilseed rape explants using sterile pipettes, 50 ml of agrobacterium solution were added and, after cautious mixing, incubated for 20 min. The agrobacteria suspension was removed, the oilseed rape explants were washed with 50 ml of callus induction medium for 1 min and then 100 ml of callus induction medium were added. The cocultivation was carried out on a rotary shaker at 100 rpm for 24 h. The cocultivation was stopped by removing the callus induction medium, and the explants were washed twice for 1 min each time with 25 ml and twice for 60 min with 100 ml each time of washing medium at 100 rpm. The washing medium with the explants was transferred into 15 cm Petri dishes, and the medium was removed using sterile pipettes. For regeneration, in each case 20-30 explants were transferred into 90 mm Petri dishes which contained 25 ml of shoot-induction medium with kanamycin.

The Petri dishes were sealed with 2 layers of Leukopor and incubated at 25 0 C and with 2000 lux in 16/8 H photoperiods. The calli which developed was transferred every 12 days to fresh Petri dishes with shoot-induction medium. All further steps for regenerating whole plants werej carried out as described by Bade, J.B. and Damm, B. (in Gene Transfer to Plants, Potrykus, I.

and Spangenberg, G.,eds, Springer Lab Manual, Springer Verlag, 1995, 30-38).

Example 8 Increasing tocopherol biosynthesis in oilseed rape The DOXS cDNA (SEQ-ID No. 1) was provided with a CaMV promoter and over-expressed in oilseed rape using the promoter. In parallel with this, the seed-specific promoter of the phaseolin gene was used in order specifically to increase the tocopherol content in the rapeseed. Oilseed rape plants transformed with the corresponding constructs were grown in a glasshouse. The a-tocopherol content of the whole plant and of the seeds of the plant was then determined. In all cases, the a-tocopherol concentration was increased by comparison with the untransformed plant.

0817/00006 Example 9 Detection of the expression of DOXS from E. coli in transgenic tobacco plants Leaf disks with a diameter of 0.9 cm were taken from completely unfolded leaves of plants containing the construct pBinAR HPPD-DOXS, and were frozen in liquid nitrogen. The leaf material was homogenized in an HEPES-KOH buffer containing proteinase inhibitors, and the protein concentration was determined from the extract using the Bio-Rad protein assay in accordance with the manufacturer's information. 45 Rg of protein from each extract were mixed with one volume of loading buffer (Laemmli, 1970) and incubated at 95 0 C for 5 min. The proteins were then fractionated on a 12.5 percent SDS-PAGE gel. The proteins were then transferred by means of semi-dry electroblots to Porablot membrane (Machery und Nagel). Detection of the DOXS protein took place using a rabbit antibody against E. coli DOXS. The color reaction is based on the binding of a secondary antibody and of an alkaline phosphatase which converts NBT/BCIP into a dye.

Secondary antibody and alkaline phosphatase were obtained from Pierce, and the procedure was in accordance with the manufacturer's information.

Figure 10 shows the detection of the DOXS protein in leaves of transgenic plants. 1: marker; 2: plant 10; 3:62; 4: 63; 5: 69; 7:71; 8:112; 9:113; 10:116; 11:WT1; 12:WT2; 13:100 ng of recombinant protein; 14:50 ng of recombinant protein; 15: 10 ng of recombinant protein.

Example Cloning of an HPPD gene from Streptomyces avermitilis U11864 Isolation of genomic DNA of the bacterium Streptomyces avermitilis U11864: A culture of Streptomyces avermitilis U11864 was grown in 300 ml of YEME medium (5 g of malt extract, 2 g of yeast extract, 2 g of glucose) at 28 0 C for 96 h. The genomic DNA of the bacterium was isolated from this culture by pelleting it initially at 5000 rev in a Sorvall RC5C fuge. The pellet was then resuspended in 1/30 of the volume of lysis buffer (25 mM EDTA, 0.5% SDS, 50 mM Tris-HCl, pH An equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) was added and incubated at 70 0 C for 10 minutes.

The aqueous phase was then separated from the phenolic in a Heraeus floor centrifuge at 3 500 rev for 15 minutes. The aqueous 0817/00006 36 supernatant was mixed with 2.5 volumes of ethanol in 1/10 volume of 8 M lithium chloride, and the nucleic acids were precipitated at room temperature for 10 minutes. The pellet was then taken up in 400 pl of TE/RNAse and incubated at 37 degrees for 10 minutes.

The solution was again shaken with one volume of phenol/chloroform/isoamyl alcohol (25:24:1), and the supernatant was precipitated with 2.5 volumes of ethanol and 1/10 volume of 8 M lithium chloride. The pellet was then washed with 80% ethanol and taken up in 400 il of TE/RNAse.

Oligonucleotides were derived for a PCR from the DNA sequence of the HPPD from Streptomyces avermitilis (Denoya et al., 1944; Acc.

Number U11864), and a BamHI restriction cleavage site was attached to the 5' end of them and an XbaI restriction cleavage site was attached at the 3' end of them. The oligonucleotide at the 5' end comprises the sequence 5'-GGATCCAGCGGACAAGCCAAC-3' (37 to 55 bases distant from the ATG in the 5' direction; in italics), and the oligonucleotide at the 3' end comprises the sequence 5'-TCTAGATTATGCCAGCCAGGCCTTG-3' (nucleotides 1845-1863 of the reverse complementary DNA sequence; in italics).

The PCR reaction was carried out with Pfu polymerase (Stratagene GmbH, Heidelberg) in accordance with the manufacturer's information. 400 ng of the genomic DNA was employed as pattern.

The PCR program was as follows: cycles: 4 sec 94 0 C, 30 sec 54 0 C, 2 min 72 0

C

cycles: 4 sec 94 0 C, 30 sec 52 0 C, 2 min 72 0

C

cycles: 4 sec 94°C, 30 sec 50 0 C, 2 min 72°C The fragment was purified by means of a Gene-Clean kit (Dianova GmbH, Hilden) and cloned in accordance with the manufacturer's information into the vector PCR-Script (Stratagene GmbH, Heidelberg). The correctness of the sequence was checked by sequencing. This revealed that the isolated gene codes for an additional amino acid. It contains the three bases TAC (coding for tyrosine) in front of nucleotide N429 in the quoted sequence (Denoya et al., 1994).

The fragment was isolated by a BamHI and XbaI digestion from the vector and ligated into a correspondingly cut Binl9AR vector downstream of the CaMV 35S promoter for expression of the gene in the cytosol. The gene was isolated as BamHI fragment from the same PCR-Script vector and was ligated into a correspondingly cut pBinl9 vector which additionally comprises the transit peptide of the potato plastidic transketolase downstream of the CaMV 0 promoter. The transit peptide ensures the plastidic localization.

0' jj VrP~~/ 0817/00006 37 The constructs are depicted in Figure 11 and 12, and the fragments have the following significance: Fragment A (529 bp) comprises the 35S promoter of cauliflower mosaic virus (nucleotides 6909 to 7437 of cauliflower mosaic virus). Fragment B (259 bp) comprises the transit peptide of transketolase. Fragment C comprises the HPPD gene. Fragment D (192 bp) comprises the polyadenylation signal of gene 3 of the T DNA of the Ti plasmid pTIACH5 (Gielen, J. et al., EMBO J. 3 (1984), 835-846) to terminate transcription.

Example 11 Production of constructs for transformation of plants with DOXS and HPPD DNA sequences To produce plants which are transgenic for DOXS and HPPD, a binary vector which contains both gene sequences was manufactured (Figure 13). The DOXS and HPPD gene sequences were each cloned as BamHI fragments as described in Example 3 and 10. The vector pBinAR-Hyg contains the 35S promoter of cauliflower mosaic virus and the polyadenylation signal of gene 3 of the T DNA of the Ti plasmid pTIACH5 (Gielen et al., 1984) for termination of transcription. The pBinAR-Hyg vector confers on plants resistance to the antibiotic hygromycin and is therefore suitable for superinfection of plants with kanamycin resistance.

To clone the HPPD in vectors which additionally contain another cDNA, oligonucleotides were derived for a PCR and had a BamHI restriction cleavage site attached at the 5' end and at the 3' end. The oligonucleotide at the 5' end comprises the sequence 5'-GGATCCTCCAGCGGACAAGCCAAC-3' (nucleotides 37 to 55 distant from the ATG in the 5' direction; in italics), and the oligonucleotide at the 3' end comprises the sequence 5'-ATGGATCCCGCGCCGCCTACAGGTTG-3' (terminating at base pair 1140 of the coding sequence, starting 8 base pairs 3' from the TAG stop codon; in italics). The PCR reaction was carried out with Tli polymerase (Promega GmbH, Mannheim) in accordance with the manufacturer's information. 10 ng of the plasmid pBinAR-HPPD were employed as template. The PCR program was as follows: cycles: 94°C 4 sec, 68 0 C 30 sec, 72 0 C 2 min cycles: 94°C 4 sec, 64°C 30 sec, 72 0 C 2 min cycles: 94 0 C 4 sec, 60 0 C 30 sec, 72 0 C 2 min 0817/00006 38 The fragment was purified using a Gene-Clean kit (Dianova GmbH, Hilden) and cloned in accordance with the manufacturer's information into the vector PCR-Script (Stratagene GmbH, Heidelberg). The correctness of the sequence was checked by sequencing. It was cut as BamHI fragment out of the vector PCR-Script and ligated into a correspondingly cut pBinAR vector which additionally contains the transit peptide of transketolase for introducing the gene product into the plastids. The result was the plasmid pBinAR-TP-HPPD (Figure 12).

For the cloning, the 35S promoter, the transketolase transit peptide, the HPPD gene and the polyadenylation signal of gene 3 of the T DNA of the Ti plasmid pTIACH5 (Gielen et al. 1984) for termination of transcription was isolated from the plasmid pBinAR-TP-HPPD by PCR. A HindIII cleavage site was attached in each case to the oligonucleotides for the promoter and the terminator. The sequence of the oligonucleotide which anneals onto the 5' region of the promoter (in italics) is 5'-ATAAGCTTCATGGAGTCAAA-GATTCAAATAGA-3', and that of the oligonucleotide which anneals onto the termination sequence (in italics) is 5'-ATAAGCTTGGACAATCAGTAAATTGAACGGAG-3'. The resulting fragment was purified using a Gene-Clean kit (Dianova GmbH, Hilden) and cloned in accordance with the manufacturer's information into the vector PCR-Script from Stratagene GmbH, Heidelberg. The correctness of the sequence was checked by sequencing. It was transferred as HindIII fragment from this PCR-Script vector into the correspondingly cut vector pBinl9 (Bevan, 1984, Nucleic Acids Res. 12, 8711-8721).

The 35S promoter, the transketolase transit peptide, the DOXS gene and the polyadenylation signal of gene 3 of the T DNA of the Ti plasmid pTIACH5 (Gielen et al., 1984) for termination of transcription was isolated by PCR from the plasmid pBinAR-TP-DOXS. An EcoRI cleavage site was attached to each of the oligonucleotides for the promoter and terminator sequence.

The sequence of the oligonucleotide which anneals onto the promoter (in italics) is 5'-ATGAATTCCATGGAGTCAAAGATTCAAATAGA-3', and that of the oligonucleotide which anneals onto the terminator sequence (in italics) is 5'-ATGAATTCGGACAATCAGTAAATTGAA-CGGAG-3'.

The fragment was purified using a Gene-Clean kit (Dianova GmbH, Hilden) and cloned in accordance with the manufacturer's information into the vector PCR-Script (Stratagene GmbH, Heidelberg). The correctness of the sequence was checked by sequencing (SEQ ID No. It was transferred as EcoRI fragment from the PCR-Script vector into the correspondingly cut vector pBinl9 (Bevan, 1984).

0817/00006 39 It was transferred as XbaI fragment from the PCR-Script vector into the correspondingly cut vector which, as described above, already contains the HPPD sequence. The result was the construct pBinAR-HPPD-DOXS (Figure 13), whose fragments have the following significance: Fragment A (529 bp) comprises the 35S promoter of the cauliflower mosaic virus (nucleotides 6909 to 7437). Fragment B comprises the transit peptide of plastidic transketolase. Fragment C comprises the HPPD gene. Fragment D comprises the polyadenylation signal of gene 3 of the T DNA of the Ti plasmid pTIACH5 (Gielen et al., 1984) for termination of transcription. Fragment E comprises the DOXS gene.

Example 12 Production of transgenic tobacco plants (Nicotiana tabacum L. cv. Samsun NN) Transgenic tobacco plants having an altered prenyl lipid content were produced by transforming tobacco leaf disks with DOXS and HPPD sequences. To transform tobacco plants, 10 ml of an Agrobacterium tumefaciens overnight culture which had grown under selection were centrifuged, the supernatant was discarded and the bacteria were resuspended in the same volume of antibiotic-free medium. Leaf disks from sterile plants (diameter about 1 cm) were bathed in this bacterial suspension in a sterile Petri dish. The leaf disks were then placed on MS medium (Murashige and Skoog, Physiol. Plant (1962) 15, 473) with 2% sucrose and 0.8% Bacto agar in Petri dishes. After incubation in the dark at 25 0 C for 2 days, they were transferred to MS medium with 100 mg/l kanamycin, 500 mg/l Claforan, 1 mg/l benzylaminopurine (BAP), 0.2 mg/l naphthylacetic acid (NAA), 1.6% glucose and 0.8% Bacto agar, and the cultivation was continued (16 hours of light/ 8 hours of dark). Growing shoots were transferred to hormone-free MS medium with 2% sucrose, 250 mg/l Claforan and 0.8% Bacto agar.

Example 13 Production of transgenic oilseed rape plants (Brassica napus) The production of transgenic oilseed rape plants having an altered prenyl lipid content was based on a protocol by Bade, J.B. and Damm, B. (in Gene Transfer to Plants, Potrykus, I.

and Spangenberg, eds, Springer Lab Manual, Springer Verlag, 0817/00006 1995, 30-38), in which the compositions of the media and buffers used are also indicated.

The transformations took place with the Agrobacterium tumefaciens strain LBA4404 (Clontech GmbH, Heidelberg). The binary vectors used were the binary constructs already described above with the total cDNAs of DOXS and HPPD. In all the binary vectors used here, the NOS terminator sequence was replaced by the polyadenylation signal of gene 3 of the T DNA of the Ti plasmid pTIACH5 (Gielen et al., 1984) for termination of transcription.

Brassica napus seeds were surface-sterilized with 70% (v/v) ethanol, washed in H 2 0 at 55 0 C for 10 min, incubated in 1% strength hypochlorite solution (25% v/v Teepol, 0.1% v/v Tween 20) for 20 min and washed six times with sterile H 2 0 for 20 min each time. The seeds were dried on filter paper for three days and 10-15 seeds were induced to germinate in a glass flask with 15 ml of germination medium. The roots and apices were removed from several seedlings (about 10 cm in size), and the remaining hypocotyls were cut into pieces about 6 mm long. The approx. 600 explants obtained in this way are washed with 50 ml of basal medium for 30 min and transferred into a 300 ml flask.

After addition of 100 ml of callus induction medium, the cultures were incubated at 100 rpm for 24 h.

An overnight culture of the agrobacterium strain was set up in Luria Broth medium with kanamycin (20 mg/l) at 290C, and 2 ml of this were incubated in 50 ml of Luria Broth medium without kanamycin at 29 0 C for 4 h until the OD 600 was 0.4-0.5. After pelleting of the culture at 2 000 rpm for 25 min, the cell pellet was resuspended in 25 ml of basal medium. The concentration of the bacteria in the solution was adjusted to an OD 600 of 0.3 by adding further basal medium.

The callus induction medium was removed from the oilseed rape explants using sterile pipettes, 50 ml of agrobacterium solution were added and, after cautious mixing, incubated for 20 min. The agrobacteria suspension was removed, the oilseed rape explants were washed with 50 ml of callus induction medium for 1 min and then 100 ml of callus induction medium were added. The cocultivation was carried out on a rotary shaker at 100 rpm for 24 h. The cocultivation was stopped by removing the callus induction medium, and the explants were washed twice for 1 min each time with 25 ml and twice for 60 min with 100 ml each time of washing medium at 100 rpm. The washing medium with the explants was transferred into 15 cm Petri dishes, and the medium was removed using sterile pipettes.

0817/00006 41 For regeneration, in each case 20-30 explants were transferred into 90 mm Petri dishes which contained 25 ml of shoot-induction medium with kanamycin. The Petri dishes were sealed with 2 layers of Leukopor and incubated at 25 0 C and with 2 000 lux in photoperiods of 16 hours of light/8 hours of dark. The calli which developed were transferred every 12 days to fresh Petri dishes with shoot-induction medium. All further steps for LL regenerating whole plants &a carried out as described by Bade, J.B. and Damm, B. (in Gene Transfer to Plants, Potrykus, I. and Spangenberg, G.,eds, Springer Lab Manual, Springer Verlag, 1995, 30-38).

Example 14 Increasing tocopherol biosynthesis in oilseed rape The cDNA of DOXS (SEQ-ID No. 3) and of HPPD (SEQ-ID No. 5) was provided with a CaMV35S promoter and overexpressed in oilseed rape using the 35S promoter. In parallel with this, the seed-specific promoter of the phaseolin gene was used in order specifically to increase the tocopherol content in the rape seed.

Oilseed rape plants transformed with the corresponding constructs were grown in a glasshouse. The a-tocopherol content of the whole plant and of the seeds of the plant was then determined. In all cases, the a-tocopherol concentration was increased by comparison with the untransformed plant.

Example Cloning of a GGPPOR gene from Arabidopsis thaliana Isolation of total RNA from completely unfolded leaves of Arabidopsis thaliana: Completely unfolded leaves of Arabidopsis thaliana were harvested and frozen in liquid nitrogen. The material was then powdered in a mortar and taken up in Z6 buffer (8 M guanidium hydrochloride, mM MES, 20 mM EDTA pH The suspension was transferred into reaction vessels and shaken with one volume of phenol/ chloroform/isoamyl alcohol 25:24:1). After centrifugation at 000 rpm for 10 minutes, the supernatant was transferred into a new reaction vessel, and the RNA was precipitated with 1/20 volumes of 1N acetic acid and 0.7 volume of ethanol (absolute). After renewed centrifugation, the pellet was first washed with 3M sodium acetate solution and, after a further centrifugation, in 70% ethanol. The pellet was then dissolved in 0817/00006 42 DEPC water, and the RNA concentration was determined by photometry.

Production of cDNA from total RNA from completely unfolded leaves of A. thaliana: ~g of total RNA were initially mixed with 3.3 il of 3M sodium acetate solution and 2 il of 1M magnesium sulfate solution and made up to a final volume of 100 Rl with DEPC water. 1 il of RNase-free DNase (Boehringer Mannheim) was added to this and incubated at 37 0 C for 45 min. After removal of the enzyme by shaking with phenol/chloroform/isoamyl alcohol, the RNA was precipitated with ethanol, and the pellet was taken up in 100 [l of DEPC water. 2.5 ig of RNA from this solution were transcribed into cDNA by means of a cDNA kit (Gibco, Life Technologies).

Oligonucleotides were derived for a PCR from the geranylgeranylpyrophosphate oxidoreductase DNA sequence (Keller et al., Eur. J.

Biochem. (1998)251(1-2), 413-417); Accession Number Y14044), and a BamHI restriction cleavage site was attached at the 5' end of these and a SalI restriction cleavage site was attached at the 3' end. The oligonucleotide at the 5' end comprises the sequence 5'-ATGGATCCATGGCGACGACGGTTACACTC-3' starting with the first codon of the cDNA (in italics), and the oligonucleotide at the 3' end comprises the sequence 5'-ATGTCGACGTGATGATAGATTACTAACAGAC-3' starting with base pair 1494 of the cDNA sequence (in italics).

The PCR reaction was carried out with Pfu polymerase from Stratagene GmbH, Heidelberg in accordance with the manufacturer's information. 1/8 of a volume of the cDNA was employed as template (equivalent to 0.3 ig of RNA). The PCR program was as follows: cycles: 94 0 C for 4 sec, 48 0 C for 30 sec, 72°C for 2 min cycles: 94°C for 4 sec, 46 0 C for 30 sec, 72 0 C for 2 min 25 cycles: 94 0 C for 4 sec, 44°C for 30 sec, 72 0 C for 2 min The fragment was purified using a Gene-Clean kit (Dianova GmbH, Hilden) and cloned in accordance with the manufacturer's information into the vector PCR-Script from Stratagene GmbH, Heidelberg. The correctness of the fragment was checked by sequencing (SEQ ID No. The gene was cloned by means of the restriction cleavage sites attached to the sequence by the primers as BamHI/SalI fragment into the correspondingly cut vector BinAR-Hyg. The latter contains the 35S promoter of cauliflower mosaic virus and the polyadenylation signal of gene 3 of the T DNA of the Ti plasmid pTIACH5 (Gielen et al., EMBO J. 3 835-846) for termination of transcription. The plasmid 0817/00006 43 confers on plants resistance to the antibiotic hygromycin and is thus suitable for superinfection of plants with kanamycin resistance. Since the plastid transit peptide of GGPPOR was also cloned, the protein ought to be transported into the plastids in transgenic plants. The construct is depicted in Figure 14. The fragments have the following significance: Fragment A (529 bp) comprises the 35S promoter of cauliflower mosaic virus (nucleotides 6909 to 7437 of cauliflower mosaic virus). Fragment D comprises the polyadenylation signal of gene 3 of the T DNA of Ti plasmid pTIACH5 (Gielen et al., 1984) for termination of transcription. Fragment F comprises the gene of GGPPOR including the intrinsic plastid transit sequence.

Example 16 Production of constructs for transformation of plants with DOXS and GGPPOR sequences To produce plants which are transgenic for DOXS and GGPPOR, a binary vector comprising both gene sequences was manufactured (Figure 15). The GGPPOR gene with the intrinsic plastidic localization sequence was cloned (as described in Example 15) as BamHI/SalI fragment into the correspondingly cut vector pBinAR-Hyg. The DOXS gene was cloned as BamHI fragment as described in Example 3. The vector pBinAR-Hyg contains the promoter of cauliflower mosaic virus and the polyadenylation signal of gene 3 of the T DNA of the Ti plasmid pTIACH5 (Gielen et al., 1984) for termination of transcription. This plasmid confers on plants resistance to the antibody hygromycin and is thus suitable for superinfection of plants with kanamycin resistance.

The 35S promoter, the transketolase transit peptide, the DOXS gene and the polyadenylation signal of gene 3 of the T DNA of the Ti plasmid pTIACH5 (Gielen et al., 1984) for termination of transcription was isolated from the plasmid pBinAR-TP-DOXS by PCR. An EcoRI cleavage site was attached in each case to the oligonucleotides for the promoter and the terminator sequence.

The sequence of the oligonucleotide which anneals onto the promoter (in italics) is 5'-ATGAATTCCATGGAGTCAAAGATTCAAATAGA-3', and that of the oligonucleotide which anneals onto the terminator sequence (in italics) is 5'-ATGAATTCGGACAATCAGTAAATTGAACGGAG-3'.

The fragment was purified using a Gene-Clean kit (Dianova GmbH, Hilden) and cloned in accordance with the manufacturer's information into the vector PCR-Script from Stratagene GmbH, Heidelberg. The correctness of the sequence was checked by 0817/00006 44 sequencing. It was transferred from the PCR-Script vector as EcoRI fragment into the correspondingly cut vector pBinl9 (Bevan, Nucleic Acids Res. 12 (1984), 8711-8721).

The 35S promoter, the GGPPOR gene and the polyadenylation signal of gene 3 of the T DNA of the Ti plasmid pTIACH5 (Gielen et al., 1984) for termination of transcription was isolated from the plasmid pBinARHyg-GGPPOR by PCR. An XbaI cleavage site was attached in each case to the oligonucleotides for the promoter and the terminator. The sequence of the oligonucleotide which anneals onto the promoter (in italics) is 5'-ATTCTAGACATGGAGTCAAA-GATTCAAATAGA-3', and that of the oligonucleotide which anneals onto the terminator sequence (in italics) is 5'-ATTCTAGAGGACAA-TCAGTAAATTGAACGGAG-3'. The fragment was purified using a Gene-Clean kit (Dianova GmbH, Hilden) and cloned in accordance with the manufacturer's information onto the vector PCR-Script from Stratagene GmbH, Heidelberg. The correctness of the sequence was checked by sequencing. It was transferred from the PCR-Script vector as XbaI fragment into the correspondingly cut vector which already contained, as described above, the DOXS sequence. The result was the construct pBinAR-DOXS-GGPPOR (Figure 15), whose fragments have the following significance: Fragment A (529 bp) comprises the 35S promoter of cauliflower mosaic virus (nucleotides 6909 to 7437 of cauliflower mosaic virus). Fragment B comprises the transit peptide of the plastidic transketolase. Fragment E comprises the DOXS gene. Fragment D comprises the polyadenylation signal of gene 3 of the T DNA of the Ti plasmid pTIACH5 (Gielen et al., 1984) for termination of transcription. Fragment F comprises the GGPPOR gene including the intrinsic plastid transit sequence.

Example 17 Production of constructs for transformation of plants with DOXS, GGPPOR and HPPD DNA sequences To produce plants which are transgenic for DOXS, GGPPOR and HPPD, a binary vector containing all three gene sequences was manufactured (Figure 16). The GGPPOR gene was provided with the intrinsic plastidic localization sequence (as described in Example 15). The pBinAR-Hyg vector used confers on plants resistance to the antibiotic hygromycin and is thus suitable for superinfection of plants with kanamycin resistance.

0817/00006 To clone HPPD into vectors which additionally contain another cDNA, oligonucleotides were derived for a PCR, and a BamHI restriction cleavage site was attached to them at the 5' end and 3' end. The oligonucleotide at the 5' end comprises the sequence 5'-GGATCCTCCAGCGGACAAGCCAAC-3' (nucleotides 37 to 55 distant from ATG in the 5' direction; in italics), and the oligonucleotide at the 3' end comprises the sequence 5'-ATGGATCCCGCGCCGCCTACAGGTTG-3' (ending with base pair 1140 of the coding sequence, starting 8 base pairs 3' of the TAG stop codon; in italics). The PCR reaction was carried out with Tli polymerase from Promega GmbH, Mannheim in accordance with the manufacturer's information. 10 ng of the plasmid pBinAR-HPPD were employed as template. The PCR program was as follows: 5 cycles: 94 0 C 4 sec, 68 0 C 30 sec, 72 0 C 2 min cycles: 94°C 4 sec, 64 0 C 30 sec, 72 0 C 2 min cycles: 94°C 4 sec, 60 0 C 30 sec, 72 0 C 2 min The fragment was purified using a Gene-Clean kit (Dianova GmbH, Hilden) and cloned in accordance with the manufacturer's information into the vector PCR-Script from Stratagene GmbH, Heidelberg. The correctness of the sequence was checked by sequencing. It was cut out of the vector PCR-Script as BamHI fragment and ligated into a correspondingly cut pBinAR vector which additionally contains the transit peptide of transketolase for introducing the gene product into plastids. The result was the plasmid pBinAR-TP-p-HPPD.

For the cloning, the 35S promoter, the transketolase transit peptide, the p-HPPD gene and the polyadenylation signal of gene 3 of the T DNA of the Ti plasmid pTIACH5 (Gielen et al. 1984) for termination of transcription was isolated from the plasmid pBinAR-TP-p-HPPD by PCR. A HindIII cleavage site was attached in each case to the oligonucleotides for the promoter and the terminator. The sequence of the oligonucleotide which anneals onto the 5' region of the promoter (in italics) is 5'-ATAAGCTTCATGGAGTCAAA-GATTCAAATAGA-3', and that of the oligonucleotide which anneals onto the termination sequence (in italics) is 5'-ATAAGCTTGGAC-AATCAGTAAATTGAACGGAG-3'. The resulting fragment was purified using a Gene-Clean kit (Dianova GmbH, Hilden) and cloned in accordance with the manufacturer's information into the vector PCR-Script from Stratagene GmbH, Heidelberg. The correctness of the sequence was checked by sequencing. It was transferred as HindIII fragment from this PCR-Script vector into the correspondingly cut vector pBinl9 (Bevan, 1984, Nucleic Acids Res. 12, 8711-8721).

0817/00006 46 The 35S promoter, the transketolase transit peptide, the DOXS gene and the polyadenylation signal of gene 3 of the T DNA of the Ti plasmid pTIACH5 (Gielen et al., 1984) for termination of transcription was isolated from the plasmid pBinAR-TP-DOXS by PCR. An EcoRI cleavage site was attached in each case to the oligonucleotides for the promoter and the terminator sequence.

The sequence of the oligonucleotide which anneals onto the promoter (in italics) is 5'-ATGAATTCCATGGAGTCAAAGATTCAAATAGA-3', and that of the oligonucleotide which anneals onto the terminator sequence (in italics) is 5'-ATGAATTCGGACAATCAGTAAATTGAACGGAG-3'.

The fragment was purified using a Gene-Clean kit (Dianova GmbH, Hilden) and cloned in accordance with the manufacturer's information into the vector PCR-Script from Stratagene GmbH, Heidelberg. The correctness of the sequence was checked by sequencing. It was transferred from the PCR-Script vector as EcoRI fragment into the correspondingly cut vector which already contained the HPPD sequence as described above.

The 35S promoter, the GGPPOR gene and the polyadenylation signal of gene 3 of the T DNA of the Ti plasmid pTIACH5 (Gielen et al., 1984) for termination of transcription was isolated from the plasmid pBinARHyg-GGPPOR by PCR. An XbaI cleavage site was attached in each case to the oligonucleotides for the promoter and the terminator. The sequence of the oligonucleotide which anneals onto the promoter (in italics) is 5'-ATTCTAGACATGGAGTCAAA-GATTCAAATAGA-3', and that of the oligonucleotide which anneals onto the terminator sequence (in italics) is 5'-ATTCTAGAGGACAA-TCAGTAAATTGAACGGAG-3'. The fragment was purified using a Gene-Clean kit (Dianova GmbH, Hilden) and cloned in accordance with the manufacturer's information into the vector PCR-Script from Stratagene GmbH, Heidelberg. The correctness of the sequence was checked by sequencing. It was transferred from the PCR-Script vector as XbaI fragment into the correspondingly cut vector which already contained the HPPD and DOXS sequences as described above. The result was the construct pBinAR-DOXS-GGPPOR-HPPD (Figure 16), whose fragments have the following significance: Fragment A (529 bp) comprises the 35S promoter of cauliflower mosaic virus (nucleotides 6909 to 7437 of cauliflower mosaic virus). Fragment B comprises the transit peptide of the plastidic transketolase. Fragment C comprises the HPPD gene. Fragment D comprises the polyadenylation signal of gene 3 of the T DNA of the Ti plasmid pTIACH5 (Gielen et al., 1984) for termination of transcription. Fragment E comprises the DOXS gene. Fragment F comprises the GGPPOR gene including the intrinsic plastid transit sequence.

Example 18 Increasing tocopherol biosynthesis in oilseed rape The cDNA of DOXS (SEQ ID No. 3) and of GGPOR (SEQ ID No. 7) was provided with a CaMV35S promoter and overexpressed in rape using the 35S promoter. In parallel with this, the seed-specific promoter of the phaseolin gene was used in order specifically to increase the tocopherol content in the rapeseed. Oilseed rape plants transformed with the corresponding constructs were grown in a glasshouse. The a-tocopherol content of the whole plant and of the seeds of the plant was then determined. In all cases, the a-tocopherol concentration was increased by comparison with the untransformed plant.

Example 19 Increasing the tocopherol biosynthesis in oilseed rape The cDNA of DOXS (SEQ ID No. of HPPD (SEQ ID No. 5) and of GGPPOR (SEQ-ID No. 7) was provided with a CaMV35S promoter and 25 overexpressed in rape using the 35S promoter. In parallel with this, the seed-specific promoter of the phaseolin gene was used in order specifically to increase the tocopherol content in the rapeseed. Oilseed rape plants transformed with the corresponding constructs were grown in a glasshouse. The a-tocopherol content of the whole plant and of the seeds of the plant was then determined. In all cases, the a--tocopherol concentration was increased by comparison with the untransformed plant.

"Comprises/comprising" when used in this specification is taken to specify 35 the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof." EDITORIAL NOTE NO 54157/99 Sequence listing pages 1-22 is part of the description.

The claims are to follow.

WO 00/08169 WO 0008169PCT/EP99/05467

SEQUENZPROTOKOLL

<110> SunGene GmnbH Co.KGaA <120> DNA-Sequenz kodierend fuer eine Synthase <130> 0050/49249 <140> 0817 00006 <141> 1999-08-04 <160> 8 <170> Patentln Vers. <210> <211> <212> <213> <220> <221> <222> 1 2458

DNA

Arabidopsis thaliana

CDS

.(2154) <400> 1 atg gct Met Ala 1 tct tot gca Ser Ser Ala 5 ttt got ttt cot Phe Ala Phe Pro tot Ser 10 tac ata ata aco aaa gga Tyr Ile Ile Thr Lys Gly gga ott tca Gly Leu Ser tct ttg gtt Ser Leu Vai act Thr gat tot tgt aaa Asp Ser Cys Lys act tct ttg tct Thr Ser Leu Ser tct aga Ser Arg aca gat ott cca Thr Asp Leu Pro tca Ser 40 oca tgt otg aaa Pro Cys Leu Lys ccc Pro aac aac aat Asn Asn Asn 144 too cat Ser His tca aao aga aga Ser Asn Arg Arg gca Al a 55 aaa gtg tgt got Lys Val Cys Ala tca Ser ott goa gag aag Leu Ala Glu Lys 192 240 ggt Gi y gaa tat tat tca Glu Tyr Tyr Ser aac As n aga oca coa act Arg Pro Pro Thr tta ott gao act Leu Leu Asp Thr aac tao ooa ato oao atg aaa aat ott tot gto aag gaa otg aaa oaa sn Tyr Pro Ile His Met Lys Asn Leu Ser Va1 Lys Glu Leu Lys Gin u1 288 WO 00/08169 WO 0008169PCT/EP99/05467 ctt tct gat Leu Ser Asp ggt gga cat Gly Gly His 115 ctg aga tca gac Leu Arg Ser Asp atc ttt aat gtg Ile Phe Asn Val tcg aaa acc Ser Lys Thr 110 act gtg gct Thr Val Ala 336 384 ttg ggg tca agt Leu Gly Ser Ser ctt Leu 120 ggt gtt gtg gag Gly Val Val Glu ctt cat Leu His 130 tac att ttc aat Tyr le Phe Asn cca caa gac aag Pro Gin Asp Lys att Ile 140 ctt tgg gat gtt Leu Trp Asp Val ggt Gi y 145 cat cag tct tat His Gin Ser Tyr cat aag att ctt His Lys Ile Leu ggg aga aga gga Gly Arg Arg Gly 432 480 528 atg cct aca atg Met Pro Thr Met caa acc aat ggt Gin Thr Asn Gly ctc Leu 170 tct ggt ttc acc Ser Gly Phe Thr aaa cga Lys Arg 175 gga gag agt Gly Giu Ser ata tct gct Ile Ser Ala 195 ga a Gi u 180 cat gat tgc ttt His Asp Cys Phe ggt Gly 185 act gga cac agc Thr Gly His Ser tca acc aca Ser Thr Thr 190 aag ggg aag Lys Gly Lys 576 624 ggt tta gga atg Gly Leu Gly Met gta gga agg gat Val Gly Arg Asp ttg Leu 205 aac aac Asn Asn 210 aat gtg gtt gct Asn Val Val Ala Att ggt gat ggt Ile Gly Asp Gly atg acg gca gga Met Thr Ala Gly gct tat gaa gcc Ala Tyr Glu Ala aac aac gcc gga Asn Asn Ala Gly tat Tyr 235 cta gac tct gat Leu Asp Ser Asp att gtg att ctt Ile Val Ile Leu aat Asn 245 gac aac aag caa Asp Asn Lys Gin gtc Val 250 tca tta cct aca Ser Leu Pro Thr gct act Ala Thr 255 ttg gat gga Leu Asp Giy agt cca cct gtt Ser Pro Pro Val ggt Gi y 265 gca ttg agc agt Ala Leu Ser Ser gct ctt agt Ala Leu Ser 270 816 cgg tta cag tct aac ccg gct ctc ,Arg Leu Gin Ser Asn Pro Ala Leu aga gag ttg aga gaa gtc gca aag Arg Giu Leu Arg Glu Val Ala Lys 864 WO 00/08169 PCT/EP99/05467 275 280 285 ggt atg Gly Met 290 aca aag caa ata Thr Lys Gin Ile gga cca atg cat Gly Pro Met His cag Gin 300 ttg gcg gct aag Leu Ala Ala Lys gta Vai 305 gat gtg tat gct Asp Val Tyr Ala cga Arg 310 gga atg ata agc Gly Met lie Ser ggt Gly 315 act gga tcg tca Thr Gly Ser Ser 912 960 1008 ttt gaa gaa ctc Phe Glu Giu Leu ggt Gly 325 ctt tac tat att Leu Tyr Tyr Ile ggt Gly 330 cca gtt gat ggg Pro Val Asp Gly cac aac His Asn 335 ata gat gat Ile Asp Asp ttg Leu 340 gta gcc att ctt Val Ala Ile Leu gaa gtt aag agt Glu Val Lys Ser acc aga acc Thr Arg Thr 350 cgt ggt tat Arg Gly Tyr 1056 aca gga Thr Gly cct tac Pro Tyr 370 cct Pro 355 gta ctt att cat Val Leu Ile His gtg acg gag aaa Vai Thr Giu Lys ggt Gly 365 gcg gag aga gct Ala Giu Arg Ala gat Asp 375 gac aaa tac cat Asp Lys Tyr His gtt gtg aaa ttt Vai Val Lys Phe 1104 1152 1200 1248 cca gca acg ggt Pro Ala Thr Gly aga Arg 390 cag ttc aaa act Gin Phe Lys Thr act Thr 395 aat gag act caa Asn Glu Thr Gin tct Ser 400 tac aca act tac Tyr Thr Thr Tyr ttt Phe 405 gcg gag gca tta Ala Glu Ala Leu gtc Val 410 gca gaa gca gag Ala Giu Ala Glu gta gac Val Asp 415 aaa gat gtg Lys Asp Vai aat ctc ttt Asn Leu Phe 435 gcg att cat gca Ala Ile His Ala atg gga ggt gga Met Gly Gly Gly acc ggg tta Thr Gly Leu 430 gta gga ata Vai Gly Ile 1296 1344 caa cgt cgc ttc Gin Arg Arg Phe aca aga tgt ttc Thr Arg Cys Phe gcg gaa Ala Glu 450 caa cac gca gtt Gin His Ala Val act Thr 455 ttt gct gcg ggt Phe Ala Ala Gly tta Leu 460 gcc tgt gaa ggc Ala Cys Giu Gly 1392 ctt aaa ccc ttc tgt gca atc tat tcg tct ttc atg cag cgt gct tat beu Lys Pro Phe Cys Ala Ile Tyr Ser Ser Phe Met Gin Arg Ala Tyr 1440 WO 00/08169 WO 0008169PCT/EP99/05467 475 gac cag gtt gtc Asp Gin Val Val cat His 485 gat gtt gat ttg Asp Val Asp Leu aaa tta ccg gtg Lys Leu Pro Val aga ttt Arg Phe 495 cat tgt His Cys 1488 1536 gca atg gat Ala Met Asp gct gga ctc gtt Ala Gly Leu Val gct gat ggt ccg Ala Asp Gly Pro gga gct Gly Ala atg gct Met Ala 530 ttC Phe 515 gat gtg aca ttt Asp Val Thr Phe atg Met 520 gct tgt ctt cct Ala Cys Leu Pro aac Asn 525 atg ata gtg Met Ile Val cca tca gat gaa Pro Ser Asp Giu gat ctc ttt aac Asp Leu Phe Asn gtt gca act gct Val Ala Thr Ala 1584 1632 1680 1728 gtt Val 545 gcg att gat gat Ala Ile Asp Asp cct tct tgt tta Pro Ser Cys Phe cgt Arg 555 tac cct aga ggt Tyr Pro Arg Gly ggt att gga gtt Gly Ile Gly Val gca Al a 565 tta cat ccc gga Leu Pro Pro Gly aac As n 570 aaa ggt gtt cca Lys Gly Val Pro att gag Ile Glu 575 att ggg aaa Ile Gly Lys ggt tat ggc Gly Tyr Gly 595 aga att tta aag Arg Ile Leu Lys gaa Glu 585 gga gag aga gtt Gly Giu Arg Vai gag ttg ttg Ala Leu Leu 590 gta atg cta Val Met Leu 1776 1824 tca gca gtt cag Ser Ala Vai Gin tgt tta gga gag Cys Leu Gly Ala gct Al a 605 gaa gaa Giu Giu 610 cga gga tta aac Arg Gly Leu Asn act gta gag gat Thr Vai Ala Asp gca Al a 620 cgg ttt tgc aag Arg Phe Cys Lys cca Pro 625 ttg gac cgt gat Leu Asp Arg Ala ctc Leu 630 att aga aga tta Ile Arg Ser Leu gat Al a 635 aag tag cac gag Lys Ser His Giu gtt Val 640 1872 1920 1968 ctg atc aag gtt Leu Ile Thr Val gaa ggt tac att Giu Gly Ser Ile gga Gi y 650 ggt ttt gga tag Gly Phe Gly Ser cac gtt His Val 655 gtt cag ttt ctt gat ata gat ggt ctt ctt gat ggc aaa ata aag tgg Vai Gin Phe Leu Ala Leu Asp Gly Leu Leu Asp Gly Lys Leu Lys Trp 2016 WO 00/08169 PTE9/56 PCT/EP99/05467 665 tac Tyr 670 gca cca gct Ala Pro Ala aga cca atg Arg Pro Met 675 gat caa cta Asp Gin Leu 690 ctg cct gat Leu Pro Asp cga Arg 680 ctc Leu att gat cac Ile Asp His ggt Gi y 685 2064 2112 gct gaa. get Ala Glu Ala atg cca tct Met Pro Ser eac atc gea His Ile Ala 700 etg ttt tga Leu Phe gea acc Ala Thr gca ctt Ala Leu 705 gagtaage tcttctaz ttactaac taaaactc taacatel aa aa aac tta. ate ggt gca cca Asn Leu Ile Gly Ala Pro 710 ~at etgttggcta aaacatatgt ~gt actgatcaga attceegec lat tgtgaagaga aaggcaaagg :tg taaaatcaat tactctettg agg gaa get krg Giu Ala 715 atacaaacac gagaagtcet caaaggttgt attgtgatga tgatcttcaa 2154 tctaaatgca accaaggtt ttggcaaeag ctatatatat gcaaagatta gtattataga gatcgtgttg taccaataac taagettgag tgacaaaaaa 2214 2274 2334 2394 2454 2458 <210> 2 <211> 717 <212> PRT <213> Arabidopsis thaliana <400> 2 Met Ala Ser Ser Ala Phe Al 1 5 Gly Leu Ser Thr Asp Ser Cy aPhe Pro Tyr Ile Ile Thr Lys Gly sLys Thr Ser 25 Pro Ser Leu Ser Ser Leu Val Ser His Ser Asp Leu Pro Ser 40 Lys Cys Leu Lys Pro Leu Ser Ser Arg Asn Asn Asn Ala Glu Lys Asn Arg Arg Gly Glu Al a 55 Arg Val Cys Ala Ser Leu Tyr Tyr Ser As n 70 Pro Pro Thr Pro 75 Leu Asp Thr Ile WO 00/08169 WO 0008169PCT/EP99/05467 Asn Tyr Pro Ile His Met Lys Asn Leu Ser Val Lys Giu Leu Lys Gin Leu Ser Asp Gly Gly His 115 Leu His Tyr Leu Arg Ser Asp Ile Phe Asn Val Gly Ser Ser Val Val Glu Leu 125 Leu Ser Lys Thr 110 Thr Vai Ala Trp Asp Val Ile Phe Asn 130 Giv His Thr 135 His Gin Asp Lys Ile 140 Gi y Gin Ser Tyr Lys Ile Leu Arg Arg Gly 145 Met Lys 160 Pro Thr Met Arg 165 His Thr Asn Gly Leu 170 Thr Gly Phe Thr Lys Arg 175 Gly Glu Ser Ile Ser Ala 195 Asn Asn Asn Asp Cys Phe Gly His Ser Leu Gly Met Gly Arg Asp Leu 205 Met Ser Thr Thr 190 Lys Giy Lys Thr Ala Giy Vai Val Ala 210 Gin Ala Val 215 Asn Gly Asp Gly Tyr Giu Ala 225 Ile Met 230 Asp Asn Ala Gly Asp Ser Asp Met 240 Val Ile Leu Asn Lys Gin Val 250 Al a Leu Pro Thr Ala Thr 255 Leu Asp Gly Arg Leu Gin 275 Gly Met Thr Pro 260 Ser Pro Pro Val Gi y 265 Arg Leu Ser Ser Asn Pro Ala Leu 280 Gi y Giu Leu Arg Glu 285 Leu Ala Leu Ser 270 Val Ala Lys Ala Ala Lys Lys Gin Ile 290 Val Asp Gi y 295 Gi y Pro Met His Gin 300 Thr Vai Tyr Ala Met Ile Ser 305 Phe Gi y 315 Gly Ser Ser Giu Glu Leu Gi y 325 Tyr Tyr Ile Gly Pro Val Asp Gly His Asn 330 335 WO 00/08169 WO 0008169PCT/EP99/05467 Ile Asp Asp Thr Gly Pro 355 Pro Tyr Ala Leu 340 Val Val. Ala Ile Leu Lys Giu Val Lys Set Thr Arg Thr Leu Ile His Val 360 Asp Val Thr Giu Lys Gl y 365 Val Arg Gly Tyr Val Lys Phe Giu Arg Ala Lys Tyr His 370 Asp~ Pro Gi y 380 Asa Ala Thr Gly 385 Tyr Arg 390 Al a Phe Lys Thr Glu Thr Gin Set 400 Thr Thr Tyr Giu Ala Leu Val 410 Met Giu Ala Glu Val Asp 415 Lys Asp Vai Asn Leu Phe 435 Ala Giu Gin Val 420 Gin Ile His Ala Giy Giy Gly Arg Arg Phe Pro 440 Phe Arg Cys Phe Asp 445 Al a Thr Gly Leu 430 Vai Gly Ile Cys Giu Gly His Ala Val Ala Ala Gly 450 Leu Lys Pro Phe Cys Tyr Set Set 465 Asp Phe 475 Lys Gin Arg Ala Tyr 480 Gin Val Val His 485 Al a Val Asp Leu Gin 490 Al a Leu Pro Val Arg Phe 495 Ala Met Asp Gly Ala Phe 515 Met Ala Pro Gly Leu Val Gi y 505 Al a Asp Gly Pro Val Thr Phe Cys Leu Pro As n 525 Val Thr His Cys 510 Met Ile Val Ala Thr Ala Set Asp Giu 530 Val Ala Al a 535 Pro Leu Phe Asn Met 540 Tyr Ile Asp Asp 545 Gi y Arg 550 Leu Set Cys Phe Pro Arg Gly Ile Gly Vai Al a 565 Pro Pro Gly Asn 570 Giy Vai Pro Ile Gly Lys Gly Arg Ile Leu Lys 580 Gly Glu Arg Val Ala Leu Leu 590 WO 00/08169 WO 0008169PCT/EP99/05467 Gly Tyr Gly 595 Ser Ala Val Gin Cys Leu Gly Ala Al a 605 Val Met Leu Glu Glu 610 Arg Gly Leu Asn Val 615 Thr Vai Ala Asp Arg Phe Cys Lys Pro 625 Leu Asp Arg Ala Leu 630 Ile Arg Ser Leu Lys Ser His Giu Val 640 Leu Ile Thr Val Glu Gly Ser Ile Gly Phe Gly Ser His Val 655 Val Gin Phe Arg Pro Met 675 Leu 660 Ala Leu Asp Gly Leu 665 Leu Asp Gly Lys Leu Lys Trp 670 Ala Pro Ala Vai Leu Pro Asp Tyr Ile Asp His Gi y 685 Asp Gin 690 Leu Ala Giu Ala Asn Leu Ile Giy 710 Gi y 695 Leu Met Pro Ser Ile Ala Ala Thr Al a '705 Leu Ala Pro Arg Giu Ala Leu Phe 715 <210> <211> <212> <213> <220> <221> <222> 3 1863

DNA

Escherichia coli

CDS

(1)..(1863) <400> 3 atg agt Met Ser 1 ttt gat att Phe Asp Ile 5 gcc aaa tac ccg Ala Lys Tyr Pro acc Thr 10 ctg gca ctg gtc gac tcc Leu Ala Leu Val Asp Ser acc cag gag Thr Gin Glu gac gaa ctg Asp Giu Leu tta Leu cga Ctg ttg ccg Arg Leu Leu Pro gag agt tta ccg Glu Ser Leu Pro ctc tgc 96 Leu Cys cgc cgc tat tta Arg Arg Tyr Leu ctc Leu 40 gac agc gtg agc Asp Ser Vai Ser c gt Arg tcc agc ggg 144 Ser Ser Gly ttc gcc tcc ggg ctg ggc acg gtc gaa ctg acc gtg gcg ctg cac 192 WO 00/08169 WO 0008169PCT/EP99/05467 His Phe Ala Ser Gly Leu Thr Val Giu Leu Thr Val Ala Leu His gtc tao aac acc Val Tyr Asn Thr cog Pro ttt gao oaa ttg Phe Asp Gin Leu tgg gat gtg ggg Trp Asp Val Gly cat His 240 288 cag got tat ccg Gin Ala Tyr Pro aaa att ttg aco Lys Ile Leu Thr gga Gi y cgc cgc gac aaa Arg Arg Asp Lys ato ggc Ile Gly aco atc cgt Thr Ile Arg ago gaa tat Ser Glu Tyr 115 cag Gin 100 aaa ggc ggt ctg Lys Giy Gly Leu cog ttc ccg tgg Pro Phe Pro Trp cgc ggc gaa Arg Gly Giu 110 tcc ato agt Ser Ile Ser 336 384 gac gta tta ago Asp Vai Leu Ser gtc Val 120 ggg oat tca tca Gly His Ser Ser aco Thr 125 goc gga Ala Gly 130 att ggt att gog Ile Gly Ile Ala got gcc gaa aaa Aia Ala Giu Lys gaa Giu 140 ggc aaa aat ogo Gly Lys Asn Arg ogc Arg 145 acc gtc tgt gtc Thr Val Cys Val ggc gat ggc gpg Gly Asp Giy Ala acc goa ggc atg Thr Aia Gly Met gcg Al a 160 ttt gaa gcg atg Phe Glu Ala Met aat Asn 165 cac gcg ggo gat His Ala Gly Asp cgt cot gat atg Arg Pro Asp Met otg gtg Leu Val 175 att oto aac Ile Leu Asn aao aao oat Asn Asn His 195 aat gaa atg tog Asn Glu Met Ser att Ile 185 too gaa aat gto Ser Giu Asn Val ggo gog cto Gly Ala Leu 190 tot toa otg Ser Ser Leu otg goa cag otg Leu Ala Gin Leu ott Leu 200 too ggt aag ott Ser Gly Lys Leu tao Tyr 205 ogo gaa Arg Glu 210 ggc ggg aaa aaa Gly Giy Lys Lys tto tot ggo gtg Phe Ser Gly Val oca att aaa gag Pro Ile Lys Giu 672 720 otg Leu 225 cto aaa ogo aco Leu Lys Arg Thr gaa Giu 230 gaa oat att aaa Glu His Ile Lys ggo Gi y 235 atg gta gtg oct Met Val Val Pro ggo Gi y 240 aog ttg tt gaa gag otg ggo ttt aao tao ato ggo cog gtg gao ggt WO 00/08169 PTE9/56 PCT/EP99/05467 Thr Leu Phe Giu Gi u 245 Leu Giy Phe Asn Tyr 250 Ile Gly Pro Val Asp Gly 255 cac gat gtg His Asp Vai aaa ggc ccg Lys Gly Pro 275 ctg Leu 260 ggg ctt atc acc Gly Leu Ile Thr cta aag aac atg Leu Lys Asn Met cgc gac ctg Arq Asp Leu 270 cgt ggt tat Arg Gly Tyr 816 864 cag ttc ctg cat Gin Phe Leu His atc Ile 280 atg acc aaa aaa Met Thr Lys Lys ggt Gi y 285 gaa ccg Giu Pro 290 gca. gaa aaa gac Aia Giu Lys Asp ccg Pro 295 atc act ttc cac Ile Thr Phe His gtg cct aaa ttt Vai Pro Lys Phe ga t Asp 305 ccc tcc agc ggt Pro Ser Ser Giy ttg ccg aaa agt Leu Pro Lys Ser ggc ggt ttg ccg Giy Giy Leu Pro agc Ser 320 912 960 1008 1056 tat tca aaa atc Tyr Ser Lys Ile ttt Phe 325 ggc gac tgg ttg Gly Asp Trp Leu tgc Cys 330 gaa acg gca gcg Giu Thr Al1a Ala aaa gac Lys Asp 335 ggc atg Gly Met aac aag ctg Asn Lys Leu gtc gag ttt Val Giu Phe 355 atg Met 340 gcg att act ccg Ala Ile Thr Pro gcg Al a 345 atg cgt gaa. ggt.

Met Arg Giu Gly tca. cgt aaa ttc ccg gat cgc tac ttc Ser Arg Lys Phe Pro Asp Arg Tyr Phe 360 gac Asp 365 gtg gca att Val Ala Ile 1104 gcc gag Ala Giu 370 caa cac gcg gtg Gin His Ala Val ttt gct gcg ggt Phe Ala Ala Gly gcg att ggt ggg Ala Ile Gly Gly tac Tyr 385 gat Asp aaa ccc att gtc Lys Pro Ile Val gcg Al a 390 att tac tcc act Ile Tyr Ser Thr ctg caa cgc gcc Leu Gin Arq Ala tat Tyr 400 1152 1200 1248 cag gtg ctg Gin Val Leu cat His 405 gac gtg gcg att Asp Vai Ala Ile ca a Gin 410 aag ctt ccg gtc Lys Leu Pro Val ctg ttc Leu Phe 415 gcc atc gac Al1a Ile Asp cgc Arg 420 gcg ggc att gtt Ala Gly Ile Val ggt Gi y 425 gct gac ggt Ala Asp Gly caa acc cat cag Gin Thr His Gin 430 1296 gct ttt gat ctc tct tac ctg cgc tgc ata ccg gaa atg qtc att 14 1344 WO 00/08169 WO 0008169PCT/EP99/05467 Gly Ala Phe 435 Asp Leu Ser Tyr Leu 440 Arg Cys Ile Pro Gi u 445 Met Val Ile atg acc Met Thr 450 ccg agc gat gaa Pro Ser Asp Glu gaa tgt cgc cag Giu Cys Arg Gin ctc tat acc ggc Leu Tyr Thr Gly tat Tyr 465 cac tat aac gat His Tyr Asn Asp ccg tca gcg gtg Pro Ser Ala Vai c gc Arg 475 tac ccg cgt ggc Tyr Pro Arg Giy 1392 1440 1488 1536 gcg gtc ggc gtg Ala Val Gly Vai ga a Gi u 485 ctg acg ccg ctg Leu Thr Pro Leu gaa Giu 490 aaa cta cca att Lys Leu Pro Ile ggc aaa Gly Lys 495 ttt ggt Phe Giy ggc att gtg Giy Ile Val acg ctg atq Thr Leu Met 515 cgt cgt ggc gag Arg Arg Giy Giu ctg gcg atc ctt Leu Ala Ile Leu cca gaa gcg gcg Pro Giu Ala Ala gtc gcc gaa tcg Vai Ala Glu Ser cig Leu 525 aac gcc acg Asn Ala Thr 1584 ctg gtc Leu Val 530 gat atg cgt ttt Asp Met Arg Phe gtg Val 535 aaa ccg ctt gat Lys Pro Leu Asp gcg tta att ctg Ala Leu Ile Leu atg gcc gcc agc Met Ala Ala Ser cat His 550 gaa gcg ctg gtc Giu Ala Leu Val acc Thr 555 gta gaa gaa aac Vai Giu Glu Asn gcc Al a 560 1632 1680 1728 att. atg ggc ggc Ile Met Gly Gly ggc agc ggc gtg Gly Ser Gly Val aac As n 570 gaa gtg ctg atg Giu Val Leu Met gcc cat Ala His 575 cgt Arg aaa cca gta Lys Pro Vai 580 ccc gtg ctg aac Pro Val Leu Asn ggc ctg ccg gac Giy Leu Pro Asp ttc ttt att Phe Phe Ile 590 ctc gat gcc Leu Asp Ala 1776 1824 ccg caa gga Pro Gin Giy 595 act cag gaa gaa Thr Gin Giu Giu atg Met 600 cgc gcc gaa ctc Arg Ala Giu Leu ggc Gly 605 gct ggt Ala Giy 610 atg gaa gcc aaa Met Giu Ala Lys atc Ile 615 aag gcc tgg ctg Lys Ala Trp Leu gca taa Al a 620 1863 WO 00/08169 WO 0008169PCT/EP99/05467 <210> 4 <211> 620 <212> PRT <213> Escherichia coi <400> 4 Met Ser Phe Asp 1 Ala Lys Tyr Pro Thr 10 Leu Ala Leu Val Asp Ser Thr Gin Giu Asp Glu Leu Leu Arg Leu Leu Pro Glu Ser Leu Pro Lys Leu Cys Ser Ser Gly Arg Arg Tyr Leu Leu 40 Asp Ser Val Ser Arg His Phe Ala Ser Gly Leu Gi y Thr Val Giu Leu Val Ala Leu His Tyr Val Tyr Asn Thr Pro Phe Asp Gin Leu Trp Asp Val Gly Gin Ala Tyr Pro Lys Ile Leu Thr Gi y Arq Arg Asp Lys Ile Gly Thr Ile Arg Ser Giu Tyr 115 Gin 100 Lys Gly Gly Leu His 105 Pro Phe Pro Trp Arg Gly Glu 110 Ser Ile Ser Asp Vai Leu Ser Giy His Ser Ser Thr 125 Ala Gly 130 Ile Gly Ile Ala Vai 135 Ala Ala Giu Lys Giu 140 Gly Lys Asn Arg Arg 145 Thr Val Cys Vai Ile 150 Gly Asp Gly Ala Thr Ala Gly Met Phe Giu Ala Met Asn 165 His Ala Giy Asp Ile 170 Arg Pro Asp Met Leu Val 175 Ile Leu Asn Asn Asn His 195 Asp 180 Asn Giu Met Ser Ser Glu Asn Val Giy Ala Leu 190 Ser Ser Leu Leu Ala Gin Leu Leu 200 Ser Gly Lys Leu Tyr 205 Arg Giu 210 Gly Gly Lys Lys Val 215 Phe Ser Gly Val Pro Ile Lys Glu WO 00/08169 PTE9/56 PCT/EP99/05467 Leu 225 Thr Leu Lys Arg Thr Giu His Ile Lys Gi y 235 Ile Met Val Val Pro Gi y 240 Leu Phe Giu Gi u 245 Gi y Gly Phe Asn Tyr 250 Leu Gly Pro Val Asp Gly 255 His Asp Vai Lys Giy Pro 275 Giu Pro Ala Leu Ile Thr Lys Asn Met Arg Asp Leu 270 Arg Gly Tyr Phe Leu His Ile 280 Ile Thr Lys Lys Gi y 285 Val Giu Lys Asp 290 Asp Pro Pro 295 Leu Thr Phe His Al a 300 Gi y Pro Lys Phe Ser Ser Giy Pro Lys Ser Giy Leu Pro 305 Tyr Ser 320 Ser Lys Ile Phe 325 Aila Asp Trp Leu Cys 330 Met Thr Ala Ala Lys Asp 335 Asn Lys Leu Vai Giu Phe 355 Ala Giu Gin Ile Thr Pro Arg Giu Gly Arg Lys Phe Arg Tyr Phe Asp 365 Aila Ser Giy Met 350 Vai Aia Ile Ile Giy Giy His Ala Vai 370 Tyr Lys Thr 375 Ile Ala Ala Giy Leu 380 Leu Pro Ile Val 385 Asp Al a 390 Asp Tyr Ser Thr Gin Arg Ala Tyr 400 Gin Val. Leu His 405 Aila Val Ala Ile Gin 410 Al a Leu Pro Val Leu Phe 415 Ala Ile Asp Gly Ala Phe 435 Met Thr Pro Arg 420 Asp Gly Ile Val Gi y 425 Arg Asp Gly Gin Leu Ser Tyr Cys Ile Pro Giu 445 Leu Thr His Gin 430 Met Vai Ile Tyr Thr Giy Ser Asp Giu Cys Arg Gin 450 Tyr His 465 Met 460 Tyr Tyr Asn Asp Gi y 470 Ser Ala Val Arg 475 Pro Arg Giy Asn 480, WO 00/08169 WO 0008169PCT/EP99/05467 Ala Val Gly Val Giu 485 Arg Leu Thr Pro Leu Gi u 490 Leu Lys Leu Pro Ile Gly Lys 495 Gly Ile Val Thr Leu Met 515 Leu Val Asp Lys 500 Pro Arg Gly Glu Lys 505 Val Ala Ile Leu Asn Phe Gly 510 Asn Ala Thr Glu Ala Ala Ala Giu Ser Leu 525 Al a Met Arg Phe 530 Glu Met Val 535 Giu Pro Leu Asp Leu Ile Leu Ala Ala Ser His 550 Gi y Ala Leu Val Thr 555 Glu Giu Glu Asn Al a 560 Met Gly Gly Al a 565 Pro Ser Gly Val Val Leu Met Ala His 575 Arg Lys Pro Pro Gin Gly 595 Ala Gly Met 610 Val 580 Thr Val Leu Asn Ile 585 Arg Leu Pro Asp Phe Phe Ile 590 Leu Asp Ala Gin Giu Glu Ala Giu Leu Gi y 605 Glu Ala Lys Ile 615 Ala Trp Leu <210> <211> 1469 <212> DNA <213> Streptornyces avermitilis <220> <221> CDS <222> (218)..(1138) <400> gatatccgag cgccgccggg tccactgcgg tccgaagccg cggatgactc cattcgactg aagccggtcg agccgcgcct gcacggtgcc gcgcgcgacc ccgagccgcc gggacatctc 120 gagcactccg atgcgcggct cccgcgccag cagcaccagg agccggccgt ccagatgatc 180 gatcgccacg gcagcccctc cagtggtcat cctgtac atg cag ccc cac gcc atg 235 Met Gin Pro His Ala Met WO 00/08169 WO 0008169PCT/EP99/05467 ggc ggt gca Gly Gly Ala ctg Leu aac aca ttg tcc Asn Thr Leu Ser ago Ser 15 gga caa goc aac Gly Gin Ala Asn tat tgc gca Tyr Cys Ala cac aca cca.

His Thr Pro 283 cot tgc Pro Cys cac tcc His Ser gga.

Gi y acg gag cga ccc Thr Giu Arg Pro cgc cat gac gca Arg His Asp Ala gac Asp cga cac cgc cog Arg His Arg Pro ggc cga coo ctt Gly Arg Pro Leu ccc Pro ggt gaa ggg aat Gly Glu Gly Asn 331 379 427 475 gga Gi y cgc ggt cgt ctt Arg Gly Arg Leu cgc Arg cgt agg caa cgc Arg Arg Gin Arg gca ggc cgc gca Ala Gly Arg Ala ctc cac cgc ctt Leu His Arg Leu cgg Arg cat gca got tgt His Ala Ala Cys ggc Gi y 80 gta ctc cgg acc Val Leu Arg Thr gga gaa Gly Glu cgg cag cog Arg Gin Pro ctt cgt cct Leu Arg Pro 105 oga Arg gac cgc ttc gta Asp Arg Phe Val cgt Arg 95 cct cac caa cgg Pro His Gin Arg ctc ggc acg Leu Gly Thr 100 ggg cca ctt Gly Pro Leu 523 571 cac ctc cgt cat His Leu Arg His gcc cgc cac coo Ala Arg His Pro cot cgc Pro Arg 120 cga cca tgt ggc Arg Pro Cys Gly gca cgg cga cgg Ala Arg Arg Arg cgt Arg 130 ogt cga cct cgc Arg Arg Pro Arg cat His 135 cga ggt ccc gga Arg Gly Pro Gly cgc Arg 140 ccg cgc cgc cca Pro Arg Arg Pro gta cgc gat cga Val Arg Asp Arg gca Al a 150 619 667 715 cgg cgc cog cto Arg Arg Pro Leu ggt Gi y 155 ogo oga goc gta Arg Arg Ala Val oga Arg 160 got gaa gga. oga Ala Glu Gly Arg gca cgg Ala Arg 165 cac ggt ogt His Gly Arg cct cgt oga Pro Arg Arg 185 ogo ogo gat ogo Arg Arg Asp Arg ota cgg oaa gao Leu Arg Gin Asp ccg oca cac Pro Pro His 180 cgg ota. ogt Arg Leu Arg 763 811 cog gac cgg ota Pro Asp Arg Leu oga Arg 190 cgq coo ota cot Arg Pro Leu Pro ccc Pro 195 WO 00/08169 WO 0008169PCT/EP99/05467 ggc cgc cgc ccc Gly Arg Arg Pro 200 cat cga cca ctg His Arg Pro Leu gat cgt cga acc qcc cgc cca ccg cac ctt cca ggc Asp Arg Arg Thr Ala Arg Pro Pro His Leu Pro Gly cgt Arg 215 ggt Gi y cgg Arg 220 caa Gin 205 caa Gin ggt Gi v 210 ccg Pro cgt cga gct cgg Arq Arg Ala Arg 225 cat ggg ctt cac His Giv Leu His gat gaa cga atg Asp Glu Arg Met 230 cat gaa gga gtt His Glu Gly Val cgg ctt cta Arg Leu Leu caa Gin 235 cat His gaa Giu 240 gac cga gta ctc Asp Arg Val Leu cgt ggg cga Arg Gly Arg cgt ggc cga Arg Gly Arg 265 cct cgc caa Pro Arg Gin cga Arg 250 c gg Arg cgc Arg ggc gct gat Giy Ala Asp 255 caa Gin 245 gtc gaa ggt Val Glu Gly 260 cga gcc cgc Arg Ala Arg gtt cta cqg Vai Leu Arg 859 907 955 1003 1051 1099 1148 cac gct caa His Ala Gin ggt Gi y 2 7 0 gtt ccc gat Val Pro Asp caa Gin 275 gga Gi y .280 cgg cgc Arg Arg 295 ggg Gi y gaa gaa gtc Giu Giu Val cgt cca gca Arg Pro Ala 300 cca gat cga Pro Asp Arg 285 cat cgc gct His Arg Ala cga Arg gaa Glu gta cct Val Pro 290 cac ggg His Gly tga catcgtcgag acggtacgca ga ca ccc tc g aagatcctcg caggaccgcc aagggcaact ctgtaggcgg cgatgcgcgc gggagtgggt cggaccgcga cgacggtctt tcaaggccct cgcggcccgg cgccggcgtc gggcgacacc cgaggacggc cttcgagatc gttcgaggcg cagttcctgg cgcgtccccg tatctgctcc atcgaacgcc atcgagcggg acacgcccga ctcgtactac tcgacaccct gcgcgagctg agatcttcac caagccggtc acggctcgat gggattcggc agcaggagaa gcggggcaac 1208 1268 1328 1388 1448 1469 <210> 6 <211> 306 <212> PRT <213> Streptoinyces avermitilis <400> 6 Met Gin Pro His Ala Met Gly Gly Ala Leu Asri Thr Leu Ser Ser Giy WO 00/08169 WO 0008169PCT/EP99/05467 1 Gin 10 Thr Ala Asn Tyr Ala Pro Cys Gl y 25 Arg Glu Arg Pro Cys Arg His Gly Arg Pro Arq Gin Arq Asp Ala Asp Leu Pro Gly His Thr Pro His His Arg Pro Al a Ar g Glu Gly Asp Gin Ala Gi y 55 Leu Gly Arg Leu Arq His Gly Arg Ala His Arg Leu Val Arg Asp Ala Ala Cys Leu Arg Thr Arg Gin Pro Arg His Arg Phe Val Arg His Gin Arg His Pro Leu 115 Arg Arg Arg Leu 100 Gi y Thr Leu Arg Leu Arg His Pro Leu Pro Pro Cys Gly Arq 125 Pro Gin Ala Arg 110 Ala Arg Arg Arg Arg Pro Arg Pro Arg 130 Ara Val His 135 Arg Gly Pro Gly Arg Asp Arg Arg Pro Leu Gl y 155 Arg Arg Ala Val Arg 160 Leu Glu Gly Arg Al a 165 Pro His Gly Arg Arg Asp Arg Arg Gin Asp Leu Pro Pro 195 Pro Pro His His Pro Arg Asp Arg Leu Leu Arg Gly Pro Asp Arg Arg 205 Gin Arg Arg Pro 190 Thr Ala Arg Arg Arg Ala Leu Pro Gly 210 Arg Pro His 215 Gi y Pro Leu Arg Arg 220 Gin Asp Glu Arg Arg Leu Leu 225 His Gly His Gly Glu His Glu Gi y 245 Glu Arg Gly Arg Arg 250 Arg Asp Arg Vai 255

GI

-VT9y Ala Asp Val Gly Arg Gly Arq Arg His Ala Gin Gly Gin Val WO 00/08169 WO 0008169PCT/EP99/05467 260 Pro Asp Gin 275 Arg Ala Arg Pro Arg 280 Gin Giu Giu Val Pro 285 Ala His 300 Asp Arg Arg Arg Ala Giu Val Pro 290 His Gly 305 Gly Vai Leu Arg Arg 295 Arg Giy Arg Pro <210> 7 <211> 1479 <212> DNA <213> Arabidopsis thaliana <220> <221> CDS <222> (1)..(1401) <400> 7 at g Met 1 gcg acg acg Ala Thr Thr aca ctc aaa tcc Thr Leu Lys Ser acc gga ctt cgt Thr Gly Leu Arg caa tca Gin Ser tca acg gag Ser Thr Giu ctc cct caa Leu Pro Gin caa Gin aca aac ttc gtc Thr Asn Phe Val cat gta ccg tca His Vai Pro Ser tca ctt tct Ser Leu Ser agg gcc act Arg Ala Thr cga cgg acc tct Arg Arg Thr Ser ctc Leu cga gta acc gca Arg Vai Thr Ala ccc aaa Pro Lys ctc tcc aac cgt Leu Ser Asn Arg aaa Lys 55 ctc cgt gtc gcc Leu Arg Vai Ala gtc Val atc ggt ggt gga Ile Gly Gly Gly gca ggc ggg gca Ala Giy Giy Ala gca gag act cta Ala Giu Thr Leu caa gga gga atc Gin Gly Gly Ile gag Gi u acg att ctc atc Thr Ile Leu Ile gag Giu cgt aag atg gac Arg Lys Met Asp aat Asn 90 tgc aag cct tgc Cys Lys Pro Cys ggt ggc Giy Giy gcg att cct ctc tgt atg gtc gga gaa ttc aac ttg ccg ttg gat att 2Rtla. Ile Pro Leu Cys Met Vai Gly Giu Phe Asn Leu Pro Leu Asp Ile 336 WO 00/08169 WO 0008169PCT/EP99/05467 100 att gat Ile Asp gct gtt Ala Val 130 cgg Arg 115 aga gtg acg aag Arg Val Thr Lys aag atg att tcg Lys Met Ile Ser tcg aac att Ser Asn Ile gat att ggt cgt Asp Ile Gly Arg ctt aag gag cat Leu Lys Giu His gag Gi u 140 tat ata ggt atg Tyr Ile Gly Met aga aga gaa gtt Arg Arg Giu Val ctt Leu 150 gat gct tat ctg Asp Ala Tyr Leu gag aga gct gag Glu Arg Ala Giu agt gga gcc act Ser Gly Ala Thr gtg Val 165 att aac ggt ctc Ile Asn Gly Leu ttc Phe 170 ctt aag atg gat Leu Lys Met Asp cat ccg His Pro 175 gag aat tgg Giu Asn Trp aaa act gga Lys Thr Gly 195 tcg ccg tac act Ser Pro Tyr Thr cat tac act gag His Tyr Thr Giu tac gat ggt Tyr Asp Gly 190 gat gct gtc Asp Ala Val gct aca ggg acg Ala Thr Gly Thr aaa aca atg gag Lys Thr Met Giu gtt Val 205 att gga Ile Gly 210 gct gat gga gct Ala Asp Gly Ala aac Asn 215 tct agg gtt gct Ser Arg Val Ala tct att gat gct Ser Ile Asp Ala ggt Gi y 225 gat tac gac tac Asp Tyr Asp Tyr gca Al a 230 att gca ttt cag Ile Ala Phe Gin agg att agg att Arg Ile Arg Ile cct Pro 240 6 72 720 768 gat gag aaa atg Asp Giu Lys Met gat gat gtg tcg Asp Asp Val Ser 260 cat gta gct gtt His Val Ala Val 275 act Thr 245 tac tat gag gat Tyr Tyr Glu Asp tta Leu 250 gct gag atg tat Ala Giu Met Tyr gtt gga Vai Gly 255 ccg gat ttc tat Pro Asp Phe Tyr ggt Gi y 265 tgg gtg ttc cct Trp Val Phe Pro aag tgc gac Lys Cys Asp 270 gac atc aag Asp Ile Lys 816 864 gga aca ggt Gly Thr Giy gtg act cac aaa Val Thr His Lys ggt Gi y 285 aag

LYS

IT',

LL,

ttc cag ctc gcg acc aga aac aga Phe Gin Leu Ala Thr Arg Asn Arg gct aag gac aag att ctt gga Ala Lys Asp Lys Ile Leu Gly WO 00/08169 PCT/EP99/05467 295 ggg Gly 305 aag atc atc cgt Lys Ile Ile Arg gag gct cat ccg Glu Ala His Pro oct gaa cat ccg Pro Giu His Pro cca cgt agg otc Pro Arg Arg Leu tat gtg act aaa Tyr Val Thr Lys 340 tcg Ser 325 aaa ogt gtg gct Lys Arg Vai Ala gta ggt gat got Val Gly Asp Ala gca ggg Ala Gly 335 960 1008 1056 tgo tot ggt gaa Cys Ser Gly Glu ggg Gly 345 ato tao ttt got got aag agt Ile Tyr Phe Ala Ala Lys Ser gga aga atg Gly Arg Met 355 tgt got gaa goo Cys Ala Giu Ala att Ile 360 gto gaa ggt toa Val Giu Gly Ser cag Gin 365 aat ggt aag Asn Gly Lys 1104 aag atg Lys Met 370 att gao gaa ggg Ile Asp Giu Gly ttg agg aag tao Leu Arg Lys Tyr ttg Leu 380 gag aaa tgg gat Glu Lys Trp Asp aca tao ttg cct Thr Tyr Leu Pro ac Thr 390 tao agg gta ott Tyr Arg Vai Leu gtg ttg oag aaa Val Leu Gin Lys gtg Val 400 1152 1200 1248 ttt tao aga toa Phe Tyr Arg Ser ccg got aga gaa Pro Ala Arg Glu gcg Ala 410 ttt gtg gag atg Phe Vai Giu Met tgt aat Cys Asn 415 gat gag tat Asp Giu Tyr gtt gcg ccg Val Ala Pro 435 gtt Va1 420 cag aag atg aca Gin Lys Met Thr gat ago tat otg Asp Ser Tyr Leu tao aag ogg Tyr Lys Arg 430 gtg aao aco Vai Asn Thr 1296 1344 ggt agt cot ttg Gly Ser Pro Leu gag Glu 440 gat ato aag ttg Asp Ile Lys Leu att gga Ile Gly 450 agt ttg gtt agg Ser Leu Val Arg aat got ota agg Asn Ala Leu Arg aga Arg 460 gag att gag aag Glu Ile Glu Lys 1392 ott agt gtt Leu Ser Vai 465 taagaaacaa ataatgaggt otatotcott tcttcatctc 1441 tatctctctt tttttgtctg ttagtaatct atctacac 1479 WO 00/08169 WO 0008169PCT/EP99/05467 <210> 8 <211> 467 <212> PRT <213> Arabidopsis thaliana <400> 8 Met 1 Ser Mla Thr Thr Thr Giu Gin Pro Gin Arg Val Thr Thr Leu Lys Ser Phe Thr Gly Leu Arg Gin Ser Asn Phe Val Val Pro Ser Leu Arg Thr Ser Ser Leu Ser Arg Ala Thr Gly Gly Gly Pro Lys Leu Leu 40 Leu Vai Thr Ala Ala Ile Ser Asn Arg Arg Val Ala Pro Ala Val Gin Gly Giy Ala Thr Al a Arg Giu Thr Leu Gly Gly Ile Ile Leu Ile Giu Cys Lys Met Asp Lys Pro Cys Gly Gly Ala Ile Pro Ile Asp Arg 115 Ala Vai Asp Met Vai Giy Giu 105 Lys Asn Leu Pro Vai Thr Lys Met Ile Ser Leu Asp Ile 110 Ser Asn Ile Ile Giy Met Ile Giy Arg 130 Val Arg Th r 135 Asp Lys Giu His Giu 140 Glu Arg Glu Vai 145 Ser Leu 150 Ile Ala Tyr Leu Arg Ala Giu Giy Ala Thr Vai 165 Ser Asn Gly Leu Lys Met Asp His Pro 175 Giu Asn Trp Lys Thr Gly 195 Ile Gly Ala T iz4 0 Pro Tyr Thr Leu 185 Lys Tyr Thr Giu Tyr Asp Gly 190 Asp Ala Vai Thr Gly Thr Thr Met Giu Vai 205 Ser Asp Gly Ala Asn 215 Arg Val Ala Ile Asp Ala WO 00/08169 WO 0008169PCT/EP99/05467 Gi y 225 Asp Tyr Asp Tyr Ile Ala Phe Gin Arg Ile Arg Ile Asp Giu Lys Met Thr 245 Tyr Tyr Giu Asp Ala Giu met Tyr Val Giy 255 Asp Asp Vai His Vai A-la 275 Pro Asp Phe Tyr Gi y 265 Trp Vai Phe Pro Lys Cys Asp 270 Asp Ile Lys Val Gly Thr Gly Thr 280 Vai Thr His Lys Gi y 285 Lys Phe 290 Gin Leu Ala Thr Arg 295 Asn Arg Ala Lys Asp 300 Lys Ile Leu Giy Lys Ile Ile Arg Giu Ala His Pro Pro Giu His Pro Pro Arg Arg Leu Lys Arg Val Ala Val Gly Asp Ala Ala Giy 335 Tyr Vai Thr Giy Arg Met 355 Lys 340 Cys Ser Gly Giu Ile Tyr Phe Ala Ala Lys Ser 350 Asn Gly Lys Cys Aia Giu Ala Val Giu Gly Ser Gin 365 Lys Met 370 Ile Asp Giu Giy Asp 375 Leu Arg Lys Tyr Leu 380 Giu Lys Trp Asp Lys 385 Thr Tyr Leu Pro Tyr Arg Vai Leu Val Leu Gin Lys Val 400 Phe Tyr Arg Ser Pro Ala Arg Giu Al a 410 Phe Val Glu Met Cys Asn 415 Asp Giu Tyr Val Ala Pro 435 Val 420 Gin Lys Met Thr Asp Ser Tyr Leu Tyr Lys Arg 430 Val Asn Thr Giy Ser Pro Leu Asp Ile Lys Leu Al1 a 445 Ile Gly 450 Ser Leu Val Arg Al a 455 Asn Ala Leu Arg Arg 460 Giu Ile Giu Lys Leu Ser Val 465 ccI

Claims (22)

1. The use of DNA sequences coding for a 1-deoxy-D-xylulose-5-phosphate synthase (DOXS) for producing plants with increased tocopherol, vitamin K, chlorophyll and/or carotenoid contents.

2. The use of a DNA sequence SEQ ID No. 1 or SEQ ID No. 3 or of a DNA sequence which hybridizes with the latter and codes for a l-deoxy-D-xylulose-5-phosphate synthase (DOXS) for producing plants with increased content of tocopherols, vitamin K, chlorophylls and/or carotenoids.

3. The use of DNA sequences coding for a synthase (DOXS) and coding for a p-hydroxyphenylpyruvate dioxygenase (HPPD) for producing plants with increased tocopherol, vitamin K, chlorophyll and/or carotenoid contents.

4. The use of a DNA sequence SEQ ID No. 1 or SEQ ID No. 3 and of a DNA sequence SEQ ID No.

5 or of a DNA sequence which hybridizes with the latter and codes for a synthase (DOXS) and a p-hydroxyphenylpyruvate dioxygenase for producing plants with increased content of tocopherols, vitamin K, chlorophylls and/or carotenoids. The use of DNA sequences coding for a l-deoxy-D-xylulose-5-phosphate synthase (DOXS) and coding for a geranylgeranyl-pyrophosphate oxidoreductase (GGPPOR) for producing plants with increased tocopherol, vitamin K, chlorophyll and/or carotenoid contents.

6. The use of a DNA sequence SEQ ID No. 1 or SEQ ID No. 3 and of a DNA sequence SEQ ID No. 7 or of a DNA sequence which hybridizes with the latter and codes for a synthase (DOXS) and a geranylgeranyl-pyrophosphate oxidoreductase (GGPPOR) for producing plants with increased tocopherol, vitamin K, chlorophyll and/or carotenoid contents.

7. The use of DNA sequences coding for a synthase (DOXS) and coding for a hydroxyphenylpyruvate dioxygenase (HPPD) and coding for a S geranylgeranyl-pyrophosphate oxidoreductase (GGPPOR) for AMENDED SHEET 0817/00006 49 producing plants with increased tocopherol, vitamin K, chlorophyll and/or carotenoid contents.

8. The use of a DNA sequence SEQ ID No. 1 or SEQ ID No. 3 and of a DNA sequence SEQ ID No. 5 and of a DNA sequence SEQ ID No. 7 or of a DNA sequence which hybridizes with the latter and codes for a l-deoxy-D-xylulose-5-phosphate synthase (DOXS), a hydroxyphenylpyruvate dioxygenase (HPPD) and a geranylgeranyl-pyrophosphate oxidoreductase (GGPPOR) for producing plants with increased content of tocopherols, vitamin K, chlorophylls and/or carotenoids.

9. A process for producing plants with increased tocopherol, vitamin K, chlorophyll and/or carotenoid contents, which comprises expressing a DNA sequence SEQ ID No. 1 or SEQ ID No. 3 or a DNA sequence which hybridizes with the latter in plants.

A process for producing plants with increased tocopherol, vitamin K, chlorophyll and/or carotenoid contents, which comprises expressing a DNA sequence SEQ ID No. 1 or SEQ ID No. 3 and a DNA sequence SEQ ID No. 5 or DNA sequences which hybridize with the latter in plants.

11. A process for producing plants with increased tocopherol, vitamin K, chlorophyll and/or carotenoid contents, which comprises expressing a DNA sequence SEQ ID No. 1 or SEQ ID No. 3 and a DNA sequence SEQ ID No. 7 or DNA sequences which hybridize with the latter in plants.

12. A process for producing plants with increased tocopherol, vitamin K, chlorophyll and/or carotenoid contents, which comprises expressing DNA sequences SEQ ID No. 1 or SEQ ID No. 3, SEQ ID No. 5 and SEQ ID No. 7 or DNA sequences which hybridize with the latter in plants.

13. A process for transforming a plant, which comprises introducing an expression cassette comprising a promoter and a DNA sequence SEQ ID No. 1 or SEQ ID No. 3 into a plant cell, into callus tissue, a whole plant or protoplasts of plant cells.

14. A process for transforming a plant, which comprises introducing an expression cassette comprising a promoter and DNA sequences SEQ ID No. 1 or SEQ ID No. 3 and SEQ ID No. AMENDED SHEET into a plant cell, into callus tissue, a whole plant or protoplasts of plant cells.

A process for transforming a plant, which comprises introducing an expression cassette comprising a promoter and DNA sequences SEQ ID No. 1 or SEQ ID No. 3 and SEQ ID No. 7 into a plant cell, into callus tissue, a whole plant or protoplasts of plant cells.

16. A process for transforming a plant, which comprises introducing an expression cassette comprising a promoter and DNA sequences SEQ ID No. 1 or SEQ ID No. 3, SEQ ID No. 5 and SEQ ID No. 7 into a plant cell, into callus tissue, a whole plant or protoplasts of plant cells.

17. A process for transforming plants as claimed in claim 13-16, wherein the transformation takes place with the aid of the strain Agrobacterium tumefaciens, of electroporation or of the particle bombardment method.

18. A plant transformed with an expression cassette as set forth in claim 13-16.

19. A plant as claimed in claim 18 selected from the group of 25 soybean, canola, barley, oats, wheat, oilseed rape, corn or sunflower.

The use of SEQ ID No. 1 or SEQ-ID No. 3 for producing a test system for identifying DOXS inhibitors

21. A test system based on the expression of an expression cassette as set forth in claim 13 for identifying DOXS inhibitors. 35

22. The use of a plant comprising a DNA sequence SEQ ID No. 1 or SEQ ID No. 3 or a DNA sequence which hybridize with the latter for producing plant and bacterial DOXS.