WO2002060939A2

WO2002060939A2 - Method for increasing the sulphur-compound content in plants

Info

Publication number: WO2002060939A2
Application number: PCT/EP2002/001131
Authority: WO
Inventors: Markus Wirtz; Oliver Berkowitz; Rüdiger HELL
Original assignee: Ipk - Institut Für Pflanzengenetik Und Kulturpflanzenforschung
Priority date: 2001-02-02
Filing date: 2002-02-04
Publication date: 2002-08-08
Also published as: WO2002060939A8; DE10104721A1; WO2002060939A3; AU2002235893A1; DE10104721B4

Abstract

The invention relates to a method for increasing the sulphur-compound content, especially the cysteine content, in plants. The inventive method comprises expression of a serin acetyl transferase gene coding for an enzymatically inactive serin acetyl transferase

Description

Process for increasing the content of sulfur compounds in plants

The present invention relates to methods for increasing the level of sulfur compounds in plants, and in particular to increasing the level of cysteine. The method according to the invention comprises the expression of a serine acetyltransferase gene which codes for an enzymatically inactive serine acetyltransferase.

The quantitatively and functionally most important reduced sulfur compounds in plants are represented by the sulfur-containing amino acid cysteine and its derivatives in a broader sense, in particular cystine, glutathione and methionine. These compounds are involved in a variety of essential functions in cells of all organisms. This also applies to taxa, which some of these sulfur compounds cannot synthesize themselves or only inadequately. B. methionine in mammals.

The biosynthesis of cysteine is catalyzed by two enzymes: serine acetyltransferase (SAT; EC 2.3.1.30) and O-acetylserine (thiol) lyase (OAS-TL; EC 4.2.99.8), the serine, acetyl-CoA and sulfide as substrates need. The

Regulation of cysteine synthesis in plants is subject to at least two mechanisms. On the one hand the feedback inhibition of the first step catalyzed by SAT through the end product cysteine, on the other hand the reversible association of the cysteine synthase complex consisting of SAT and OAS-TL.

So far, active SAT has only been found in complex with OAS-TL, while OAS-TL is inactivated by binding SAT. The cysteine synthase complex therefore consists of active SAT and inactive OAS-TL. The reaction intermediate O-acetylserine then diffuses out of the complex and is converted into sulfate to cysteine by excess and free and active OAS-TL dimers. Sulfide stabilizes the cysteine synthase complex, whereas the intermediate O-acetylserine (OAS) dissociates the complex. This is believed to be a protein interaction system for metabolic control of the rate of cysteine synthesis realized in which the SAT activity has a limiting effect. Indeed, overexpression of SAT in transgenic plants causes an increase in cysteine and glutathione levels.

Cysteine synthesis in plants takes place in the cytosol, in the plastids and in the mitochondria. Glutathione is formed in the cytosol and plastids as a transient storage of cysteine in the cell and has extensive functions in the stress resistance of plants. Increasing the content of sulfur compounds in plants is of great biotechnological interest. A higher content of sulfur compounds leads to an improvement in the nutritional value of useful plants for human and animal nutrition. But irrespective of whether the crops are used by animals or humans, an increase in the level of sulfur compounds is useful for the plant itself, since this improves the resistance or tolerance of plants to biotic and abiotic stress factors, since many protective mechanisms depend on availability depend on reduced sulfur compounds. Examples include the detoxification of heavy metals, xenobiotics including herbicides, radical compounds including those of oxygen, and the defense against phytopathogens, especially fungi.

The main source of cysteine and methionine in human and animal food are plants which, as described above, can form all other sulfur compounds from cysteine. These compounds are involved in a variety of essential functions in cells of all organisms. The supply of these ingredients is important for optimal growth and health for organisms such as mammals, which can not or only insufficiently cover this need themselves. Cysteine synthesis is therefore the appropriate starting point for improving the levels of sulfur compounds in crop plants. The cysteine synthase complex forms a reversible protein-protein interaction system for the metabolic control of the cysteine synthesis rate. An intervention in this The regulatory mechanism therefore has a greater effect on the formation of cysteine than the direct increase in the levels of the enzymes involved in transgenic plants.

The economic interest therefore concerns bio and agrotechnological areas such as phytoremediation, crop protection and yield and quality breeding.

Surprisingly, it has now been possible to provide a new method for increasing the content of sulfur compounds, in particular cysteine, in plants, in which a serine acetyltransferase enzyme which is enzymatically inactivated by mutation is expressed in plants. Although the serine acetyltransferase does not itself show any enzymatic activity, it is nevertheless able to interact with the O-acetylserine (thiol) lyase (OAS-TL).

The method according to the invention is thus based on the targeted change in the regulation of the cysteine-synthase complex in transgenic plants. In principle, this change is independent of the interventions in cysteine biosynthesis described so far.

In the context of this invention, “enzymatically inactive” means that, in a conventional enzyme test, for example carried out with crude extracts from cells which have been cultured in full medium, the SAT enzyme activity is markedly reduced compared to the wild type protein, which as a positive control is generally under the conditions mentioned shows an enzyme activity of at least approximately 1.0 nmol OAS / min × μg protein. “Enzymatically inactive” preferably means one compared to the wild type protein by at least 5 times, in particular by at least 10 times and particularly preferably by at least 15 times, 20 -foldly reduced enzyme activity. Most preferably, "enzymatically inactive" means that in a standard enzyme test, as in the present application with reference to Kredich and Becker (1971, In Methods in Enzymology (Tabor and Tabor, eds), pages 459-469, Academic Press, New York, USA), no SAT enzyme activity is detectable, that is, the activity is below the detection limit, while the wild type protein as a positive control very clearly shows activity.

In any case, the check whether a SAT protein is to be regarded as "enzymatically inactive" in the sense of the invention should always be carried out in comparison with the wild type protein. For example, for a "enzymatically inactive" SAT protein in the sense of the invention Activity can be detectable if the protein has been heavily purified and used in large quantities in the enzyme test. In comparison to wild type protein of similar purity and in similar amounts, the SAT protein according to the invention would only have an activity of at most 20%, preferably at most 10%, particularly preferably at most 5% and most preferably at most 3%, 2%, 1 %. In addition to the degree of purity of the protein, the number of plasmid copies, the type of growth medium, the culture conditions and other factors can of course also influence the SAT activity measured in the enzyme test. In view of these factors too, the activity should always be determined in comparison to wild-type protein or another SAT protein with significant enzyme activity.

Whether the feature "enzymatically inactive" in the sense of the invention is fulfilled for a SAT protein can also be seen from the fact that the SAT enzyme is not able to functionally cysteine auxotrophic E. cob ^' mutant (cysK) Cysteine-deficient bacterial strains are described in the present application, but are also known in the prior art, for example the EC1801 strain, which can be obtained from the E. coli Genetic Stock Center, Yale University, New Haven, CT, USA. The functional complementation can be carried out according to conventional protocols If the activity of a SAT protein is not sufficient to perform the necessary biological function in a complementation test (eg in minimal medium, without cysteine), it is in any case an "enzymatically inactive""SAT protein in the sense of the present invention. This means that in the total protein extract of the transformed cysE strain, as described above, no enzymatic SAT activity can be detected or is below the detection limit. In this case, too, a comparison with a wild type protein or another SAT protein with clear activity 5 is advisable.

The invention thus relates to a method for increasing the content of sulfur compounds, in particular cysteine in plants, characterized by the transfer and expression of a serine acetyltransferase gene which codes for an enzymatically inactive serine acetyltransferase.

The increase in the cysteine content subsequently leads to an increase in the levels of glutathione and methionine. The invention thus also relates to a method for increasing the content of glutathione and / or methionine in plants. L5

In a preferred embodiment, the enzymatically inactive serine acetyltransferase is able to interact with OAS-TL.

Without wishing to be bound by a hypothesis, it is currently assumed that SAT and the enzyme OAS-TL following in the synthetic route are active in corresponding isoforms in cytosol, plastids and mitochondria of plants (Lunn et al. (1990) Plant Physiol. 94, 1345-1352; Rolland et al. (1992) Plant Physiol. 98, 927-935; Noji et al. (1998) J. Biol. Chem. 273, 32739-32745). A disruption of cysteine synthesis in one compartment can therefore potentially be compensated for or overcompensated by the other compartments. An enzymatically inactive SAT protein in the sense of the invention is de facto enzymatically inactive, but can still interact with the second enzyme OAS-TL and form the cysteine-synthase complex. Hell and Hillebrand (2001, Curr. 30 Op. Biotechnol. 12, 161-168) provide a hypothetical model of the cysteine-synthase complex. The dominant-negative mutations of components of the cysteine synthase complex result in a multiple increase in the total contents of soluble cysteine, glutathione and methionine. The accumulation observed so far in transgenic plants amounts to up to 3 ox for cysteine, 8x for glutathione and 2x for methionine. However, the underlying synthetic capacity is even higher and can therefore not only be used for the formation of sulfur-containing primary and secondary substances, but also sulfur-rich storage proteins.

Applications are seen in improving the nutritional value of crops for human and animal nutrition. Furthermore, the resistance or tolerance of plants to biotic and abiotic stress factors can be improved, since many protective mechanisms depend on the availability of reduced sulfur compounds. Examples include the detoxification of heavy metals, xenobiotics including herbicides, radical compounds including oxygen and the defense against phytopathogens, especially fungi.

The serine acetyltransferase expressed in plants is preferred due to at least one mutation, in particular at least one amino acid exchange, within the amino acid motif preserved in SAT enzymes

GKX] X ₂ GDRHPKIGD

inactivated. The amino acid X is any amino acid; X ₁ is preferably Q or A; the amino acid X ₂ is preferably C or S. The amino acids located N- or C-terminal next to this motif are also highly conserved in serine acetyltransferases. The core motif within the amino acid sequence motif mentioned is DR H. An amino acid exchange within this core motif is particularly preferred. In a particularly preferred embodiment, the mutation which leads to the enzymatic inactivation of the SAT is an amino acid exchange of the amino acid histidine within the motif mentioned. An exchange of histidine to alanine is particularly preferred.

In the attached Figure 1 different SAT amino acid sequences are shown in comparison. SAT1 stands for Arabidopsis thaliana SAT isoform A (cDNA SAT-1, database axcession no. U 22964), SAT5 stands for Arabidopsis thaliana SAT isoform B (cDNA SAT-5, database axcession no. Z 34888), p AT52 stands for the Arabidopsis thaliana SAT isoform C (cDNA SAT-52, database axcession no. U 30298), CysE stands for the SAT enzyme from S. typhimurium (CysE, database accession no. A 00198); TDT stands for tetrahydrodipicolinate-N-succinyl transferase from E. coli (TDT; database accession no. P 56220); LpxA stands for UDP-N-acetylglucosamine acyltransferase from E. coli (LpxA; database accession no. P 10440). Further sequence information can be found in Murillo et al. (1995) Cell. Mol. Biol. Res. 41, 425-433; Howarth et al. (1997) Biochim. Biophys. Acta 1350, 123-127; Saito et al. (1995) J. Biol. Chem. 270, 16321-16326, Genbank Accession no. D 88530 (K. Saito). The enclosed alignment can be used to determine the position of the motif suitable for inactivating the SAT enzyme or to determine the position of the conserved amino acid motif in further SAT enzymes which can be found in the prior art. For example, the core motif D R H is in the Arabidopsis thaliana SAT isoform A at amino acid 307-309, whereby the numbering always refers to the first methionine of the longest open reading frame. The position of the motif in the other SAT isoforms can easily be found in the amino acid alignment shown in Fig. 1.

In addition to the SAT genes mentioned above, the person skilled in the art has other SAT sequences described in the prior art and available in the gene databases available that are suitable for the implementation of the invention. In addition, the person skilled in the art is easily able to isolate further SAT gene sequences from any organism using routine methods such as PCR or screening libraries with suitable SAT gene probes. Of course, the method according to the invention can also be implemented with SAT sequences which will be isolated in future and published in the database. The person skilled in the art is also able without difficulty to incorporate sequences that will be available in the future into an alignment as shown in FIG. 1 and to determine the position of the sequence motifs mentioned above.

The transfer and expression of a nucleic acid sequence which codes for an enzymatically inactive but still capable of interacting with OAS-TL in the cytosol or in the plastids results in a multiple increase in the total contents of cysteine, glutathione and z. T. of methionine in plants. The novelty of this method, which causes an increase in the cysteine content in plants and consequently an increase in the content of glutathione and methionine, consists in the completely unexpected increase in the content of sulfur compounds by expression of an enzymatically inactive protein. An enzymatically inactive SAT protein can not only be produced by means of mutagenesis, but the serine acetyltransferase can also be achieved by inhibition by suitable ligands or proteins, including antibodies.

The methods and molecular biological techniques required to implement the method according to the invention for increasing the content of sulfur-containing compounds in plants are well known to the person skilled in the art, for example, he can generate mutations by means of commercially available mutagenesis kits. The person skilled in the art can also derive the relevant techniques from the standard work of Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA. The person skilled in the art can also determine by mutations in the above-mentioned amino acid motif which mutation, ie at which position and which amino acid exchange (ie i) which amino acid and ii) which amino acid is to be exchanged, at the same time inactivating the S AT- Enzyme and an increase in the content of sulfur-containing compounds in plant cells leads. For this purpose, the mutation constructs must either be transferred to plant cells and the effects of expression in plant cells tested, or the enzyme activity or the ability to form complexes with OAS-TL must be tested using the methods described below. The person skilled in the art can also easily check whether a particular mutation leads to the desired inactivation of the SAT enzyme, as can be seen from the examples below, by transferring and expressing a plasmid containing the mutated SAT gene in bacterial strains. who have SAT deficiency.

The various methods of transferring gene constructs and planting plasmids are also well known to those skilled in the art. The same applies to regulatory sequences that are suitable for the expression of genes in plants, such as. B. constitutive promoters, tissue and development-specific promoters, inducible promoters, enhancer sequences and other regulatory

Sequences that control the transcription and translation of a coding DNA sequence operatively linked to a promoter region in transformed plant cells.

To prepare the introduction of foreign genes into higher plants or their cells, a large number of cloning vectors are available which contain a replication signal for E. coli and a marker gene for the selection of transformed bacterial cells. Examples of such vectors are pBR322, pUC series, M13mp series, pACYC184 etc. The desired sequence, in the present case a mutated SAT gene, can be found at a suitable restriction site in - lü ¬

the vector will be introduced. The plasmid obtained is then used for the transformation of E. co / t ^' cells. Transformed E. cob ^' cells are grown in an appropriate medium and then harvested and lysed, and the plasmid is recovered. Restriction analyzes, gel electrophoresis and other biochemical-molecular biological methods are generally used as the analytical method for characterizing the plasmid DNA obtained. After each manipulation, the plasmid DNA can be cleaved and DNA fragments obtained can be linked to other DNA sequences.

A large number of techniques are available for introducing DNA into a plant host cell, and the person skilled in the art can determine the appropriate method in each case without difficulty. These techniques include the transformation of plant cells with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as the transformation medium, the fusion of protoplasts, injection, electroporation, the direct gene transfer of isolated DNA to protoplasts, the introduction of DNA using the biolistic method and further possibilities that have been well established for several years and belong to the usual repertoire of the specialist in plant molecular biology or plant biotechnology.

When injecting and electroporation of DNA into plant cells, there are no special requirements per se for the plasmids used. The same applies to direct gene transfer. Simple plasmids such as e.g. B. pUC derivatives can be used. However, if whole plants are to be regenerated from such transformed cells, the presence of a selectable marker gene is recommended. The usual selection markers are known to the person skilled in the art and it is not a problem for him to select a suitable marker.

Depending on the method of introducing desired genes into the plant cell, additional DNA sequences may be required. Are z. B. for the transformation of If the plant cell uses the Ti or Ri plasmid, at least the right boundary, but often the right and left boundary of the T-DNA contained in the Ti or Ri plasmid, must be connected as a flank region to the genes to be introduced. If Agrobacteria are used for the transformation, the DNA to be introduced must be cloned into special plasmids, either in an intermediate or in a binary vector. The intermediate vectors can be integrated into the Ti or Ri plasmid of the agrobacteria on the basis of sequences which are homologous to sequences in the T-DNA by homologous recombination. This also contains the vir region necessary for the transfer of the T-DNA. Intermediate vectors cannot replicate in agrobacteria. Using a helper plasmid, the intermediate vector can be transferred to Agrobacterium tumefaciens (conjugation). Binary vectors can replicate in E. coli as well as in Agrobacteria. They contain a selection marker gene and a linker or polylinker, which are framed by the right and left T-DNA border region. They can be transformed directly into the agrobacteria. The agrobacterium serving as the host cell is said to contain a plasmid which carries a vir region. The vir region is necessary for the transfer of the T-DNA into the plant cell. Additional T-DNA may be present. The agrobacterium transformed in this way is used to transform plant cells. The use of T-DNA for the transformation of plant cells has been intensively investigated and has been sufficiently described in well-known overview articles and manuals for plant transformation. For the transfer of the DNA into the plant cell, plant explants can expediently be cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes. From the infected plant material (e.g. pieces of leaf,

Stem segments, roots, but also protoplasts or suspension-cultivated plant cells) can then regenerate whole plants again in a suitable medium, which can contain antibiotics or biocides for the selection of transformed cells. Once the introduced DNA is integrated in the genome of the plant cell, it is generally stable there and is also retained in the progeny of the originally transformed cell. It normally contains a selection marker which shows the transformed plant cells resistance to a biocide or an antibiotic such as kanamycin, G 418, bleomycin, hygromycin, methotrexate,

Glyphosate, streptomycin, sulfonylurea, gentamycin or phosphinotricin u. a. taught. The individually selected marker should therefore allow the selection of transformed cells from cells that lack the inserted DNA. Alternative markers are also suitable for this, such as nutritive markers and screening markers (such as GFP, green fluorescent protein). Of course, it can also be completely open

Selection markers are dispensed with, which, however, goes hand in hand with a rather high need for screening. If marker-free transgenic plants are desired, strategies are available to the person skilled in the art which permit subsequent removal of the marker gene, e.g. B. cotransformation, sequence-specific recombinases.

The regeneration of the transgenic plants from transgenic plant cells is carried out according to customary regeneration methods using known nutrient media. The plants obtained in this way can then be examined using conventional methods, including molecular biological methods, such as PCR, blot analyzes, for the presence of the introduced nucleic acid which encodes an enzymatically inactive SAT.

The transgenic plant or the transgenic plant cells can be any monocot or dicot plant or plant cell. They are preferably useful plants or cells of useful plants. It is particularly preferably barley, wheat, rye, oats, corn, rice, potatoes, soybeans, lupine, peas, broad beans. In particular, members of the legumes are characteristically poor in S-containing amino acids and are therefore particularly suitable for increasing the cysteine content. Because of targeted Mixtures of cereals, maize and legumes in animal feed are generally such an improvement. In principle, any crop that is suitable for nutritional purposes is desirable for the implementation of the invention. This also results in the above-mentioned advantages of increased levels of sulfur-containing compounds in plants.

In addition, it should be noted that the improved nutritional value due to increased thiol levels in plants affects all parts and organs of plants that are used for human and animal nutrition, including seeds, leaves, tubers etc.); see e.g. Schnug (1997) In Sulfur Metabolism in higher plants; Pages 109-130, WJ. Cram et al., Eds, Backhuys Publ., Leiden, NL; Herbers and Sonnewald (1999) Curr. Opin. Biotech. 10, 163-168; Tabe and Higgins (1998) TIBS 7, 282-286).

Since increasing the content of sulfur-containing compounds also benefits plant health, any crop is of interest anyway. Tolerance to biotic and abiotic stress is often mediated by glutahione, the immediate precursor of which is cysteine; see, for example, in connection with resistance to pathogens Bohlmann H. (1993) In Sulfur Nutrition and Assimilation in Higher Plants, pages 211-219, De Kok et al., eds, SPB Academic Publ., The Hague; in connection with resistance to xenobiotics and herbicides Lamoureux and Rusness (1993) In Sulfur Nutrition and Assimilation in Higher Plants, pages 221-237, De Kok et al., eds, SPB Academic Publ., The Hague; in connection with heavy metal resistance Rauser (1993) In Sulfur Nutrition and Assimilation in Higher Plants, pages 239-251, De Kok et al., eds, SPB Academic Publ., The Hague; in connection with the tolerance to gaseous pollutants such as H ₂ S, SO Ernst (1993) In Sulfur Nutrition and Assimilation in Higher Plants, pages 295-314, De Kok et al., eds, SPB Academic Publ., The Hague; and in connection with tolerance to oxidative stress: Stulen and De Kok (1993) In Sulfur Nutrition and Assimilation in Higher Plants, pages 125-138, De Kok et al., eds, SPB Academic Publ, The Hague; In view of the increase in the glutathione content in the transgenic plants achieved by the method according to the invention and the associated increase in stress resistance, the invention also relates to a method for increasing the stress resistance in transgenic plants by increasing the glutathione content in the plants by means of expression of an enzymatically inactive SAT.

The invention also relates to propagation material and harvest products of the plants according to the invention, for example fruits, seeds, tubers, rhizomes, seedlings, cuttings, etc.

Since the increase in the content of sulfur compounds, especially cysteine, in the transgenic plants i.a. can be used to improve the nutrition of humans and animals, the invention naturally relates in particular to those parts, fruits, extracts and products obtained from plants or fruits and parts of plants which can serve as food for humans and animals.

The expression of the inactive SAT in the plants or plant cells can be detected and tracked using conventional molecular biological and biochemical methods. These techniques are known to the person skilled in the art and are easily able to choose a suitable detection method, for example a Northern blot analysis for the detection of SAT-specific RNA or for determining the amount of accumulation of SAT-specific RNA or a Southern Blot analysis to identify DNA sequences coding for SAT.

The invention thus also relates to a method for producing plants with a content of sulfur-containing compounds which is higher than that of wild-type plants, comprising the following steps: a) the transfer to plant cells of a recombinant nucleic acid molecule or vector which contains a DNA sequence which codes for an enzymatically inactive SAT which is able to interact with OAS-TL; b) the regeneration of plants from the transformed plant cells.

The present invention is illustrated in the following examples, which serve only to illustrate the invention and are in no way to be understood as a limitation.

Examples

General cloning procedures

Cloning procedures, such as B. restriction cleavage, DNA isodization, agarose, gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linking of DNA fragments, transformation of E. coli cells, cultivation of bacteria, sequence analysis of recombinant DNA, were according to Sambrook et al. (1989) vide supra. The transformation of Agrobacterium tumefaciens was carried out according to the method of Höfgen and Willmitzer (Nucl. Acids Res. (1988) 16, 9877). The agrobacteria were grown in YEB medium (Vervliet et al. 1975 J. Gen. Virol. 26, 33).

Bacterial strains and plasmids

E. coli (XL 1 Blue) bacteria were purchased from Stratagene, La Jolla, USA. The agrobacterial strain used for plum transformation (C58C1 with the plasmid pGV 3850kan) was developed by Deblare et al. (1985, Nucl. Acids Res. 13, 4777). The vector Bin19 or its derivatives pBinAR and pBinAR-TkTp (Bevan (1984) Nucl. Acids Res. 12, 8711-8720) were used for cloning.

plant transformation

To transform tobacco plants (Nicotiana tabacum L. cv. Samsun NN), 10 ml of an overnight culture grown under selection from

Centrifuged Agrobacterium tumefaciens, discarded the supernatant and resuspended the bacteria in the same volume of antibiotic-free medium. Leaf disks of sterile plants (diameter approx. 1 cm) were bathed in this bacterial solution in a sterile petri dish. The leaf disks were then placed in Petri dishes on MS medium (Murashige and Skoog (1962) Physiol. Plant 15, 473) with 2% sucrose and 0.8% Bacto agar. After two days incubation in the dark at 25 ° C, they were on MS medium with 100 mg / 1 kanamycin, 500 mg / 1 claforan (cefotaxime), 1 mg / 1 benzylaminopurine (BAP), 0.2 mg / 1 naphthylacetic acid (NAA) , 1.6% glucose and 0.8% Bacto agar and cultivation (16 hours light / 8 hours dark) continued. Growing shoots were transferred to hormone-free MS medium with 2% sucrose, 250 mg / 1 Claforan and 0.8% Bacto agar.

Production of an enzymatically inactive serine acetyltransferase (SAT) which is still able to interact with OAS-TL

The SAT-A from Arabidopsis thaliana described in the prior art with regard to its amino acid sequence and the underlying DNA sequence was converted to alanine by directed mutagenesis of the amino acid histidine 309 disabled (numbering refers to the first methionine in the open reading frame). The directed mutagenesis of the associated cDNA in the plasmid pBlueScript (Stratagene) was carried out by base pair exchange using a commercially available method from Promega (Heidelberg, Germany).

Site-directed mutagenesis of the SAT-A cDNA was carried out with pBS / ΔS AT1-6 (Bogdanova et al. (1995) FEBS Lett. 358, 43-47). The Promega GeneEditor in vitro Site Directed Mutagenesis System used achieved an average of 80% positive clones. The point mutations were verified by DNA sequencing and the resulting amino acid changes were numbered in relation to the start codon of the longest possible open reading frame of a mitochondrial SAT-A cDNA (cDNA SAT-1 (Roberts et al. (1996) Plant Mol. Biol. 30, 1041-1049)).

The inactivation of the SAT mutant by the amino acid exchange in position 309 (histidine -> alanine) was due to the lack of heterologous complementation of a S AT-free E. cob ^' mutant and confirmed by enzyme determination in vitro (maximum 1% residual activity). The fractions with O-acetylserine (thiol) lyase (OAL-TL) were demonstrated by heterologous expression in the yeast "two-hybrid" system and co-expression in E. coli with subsequent biochemical purification.

The inactivation of the E. coli cysE gene to generate a SAT-free E. coli mutant was carried out according to the method described by Hamilton et al. described method carried out (Hamilton (1989) J. Bacteriol. 171, 4617-4622). This should provide a bacterial strain that is more stable in terms of its SAT deficiency than that currently available in the prior art. For this purpose, the wild-type cysE gene was cloned by PCR and inactivated by inserting a gentamycin resistance cassette into a Clal restriction site at position 522, based on the start codon of the c sE gene. After cloning this cassette into the plasmid pMAK705, which has a temperature-sensitive origin of replication the inactivated cysE gene was integrated into the genome of E. coli C600 via homologous recombination to give the strain MW1 (tbr, leu, thi, lac, l-Pl + F ', cysE, Gm ^r ). Complementaxion tests using the E. co / z strains ΕC1801 (E. coli Genetics Stock Center, Yale University, New Haven, CT, USA) or MW1 were carried out on M9 minimal medium agar plates with or without cysteine with the addition of induction agents and selective antibiotics.

The constructs for the expression of the mitochondrial SAT-A from Arabidopsis thaliana included BS / ΔSATl-6 (X82888 (Bogdanova et al. (1985) vide supra)), pET / ΔSATl-6 and mutated forms of SAT-A. In order to obtain the latter plasmid, the coding region of the SAT-A was amplified by PCR from base pair 28-939 without a mitochondrial transit peptide using specific primers, flanked by EcoRI and Xhol cleavage sites. This fragment was cloned into the plasmid pCAP (Röche, Mannheim, Germany), the sequence was verified by sequencing both strands and inserted into the corresponding cleavage sites of pET29a (Novagen, Madison, USA), which resulted in a fusion protein with the 35 amino acids of the S- Day at its N-terminus for affinity purification on S-agarose resulted. Mitochondrial OAS-TL from A. thaliana was similarly obtained by cloning a PCR product that contained the mature protein without the mitochondrial transit peptide from base pair 172-1162 (AJ271727 (Hesse et al. (1999) Amino Acids 16, 113-131) into the Ncol-BamHI cleavage sites of pET3d, which gave pET / OAS-C.

The expression, cultivation and affinity purification of SAT and OAS-TL using the S-tag system (Novagen) were essentially as described by Droux et al. (1998, Eur. J. Biochem. 255,235-245), with the following modifications. After the last washing step, the S-tag was not removed by proteolytic cleavage on the affinity column, since this treatment resulted in fractions with labile SAT activity. Instead, SAT with 3 M MgCl ₂ , which was then removed by gel filtration on PDIO columns (Amerschan, Freiburg, Germany). In vitro interaction of SAT and OAS-TL on the column was determined in a standard washing and elution protocol with or without 1 mM OAS (O-acetylserine) as described by Droux et al. (1998, vide supra).

The determination of the protein concentration and the separation of proteins were carried out according to standard protocols (e.g. Sambrook et al. (1989) vide supra).

The SAT enzyme activity with and without OAS-TL was determined in a standard assay based on the method of Kredich and Becker (1971, In Methods in Enzymology (Tabor and Tabor, eds), pages 459-469, Academic Press, New York , USA). Raw or purified recombinant SAT protein was in a volume of 250 ul (50 mM Tris / HCl, pH 7.5, 0.2 mM acetyl-CoA, 2mM

Dithiotlireitol, 5mM serine) incubated at 25 ° C and A ₂₃₂ was taken up to 3 min.

The OAS-TL activity was examined under saturated conditions as described above (Nakamura et al. (1987) Plant Cell Physiol. 28, 885-891). kinetic

Analyzes were carried out with the SigmaPlot software, which allowed hyperbolic adjustments based on the Michaelis-Menten equation: v = V _ma χ x [S]) / (K _m + [S]).

For the interaction analyzes using the yeast "two-hybrid" system, the transformation of the yeast strains HF7c and PCY2, the selection on minimal medium and β-galactosidase assays were carried out as already described (Bogdanova and Hell (1997) Plant J. 11, 251 -262). PCR with specific primer pairs, flanked by Sall or Spel interfaces, was used for all constructs to convert the coding regions into the corresponding ones Insert restriction sites of pPC86 (GAL4 activation domain) and pPC97 (GAL4 DNA binding domain) (Chevray and Nathans (1992) Proc. Natl. Acad. Sci. USA 89, 5789-5793). EST 181H17T7 (GenBank Accession Number AJ2711727) was used as a template to generate OAS-TL C without a mitochondrial transit peptide from base pair 172-1162. In contrast to the fill length construct previously used (Bogdanova et al. (1997) vide supra), the pPC vectors with mitochondrial SAT-A without a mitochondrial transit peptide were constructed by amplifying base pairs 28-939 (X82888 (Bogdanova et al. ( 1995) vide supra).

Expression of the inactive SAT mutant (H309A) in plants

The cDNA of the SAT mutant was in the binary transformation vector pBin-AR (Höfgen and Willmitzer (1990) Plant Sei. 66, 221; pBinl 9-Ursprung) or in its derivative pBinAR-TkTp (R. Badur, dissertation, University of Göttingen , 1998) cloned. The 35S RNA promoter of the Cauliflower Mosaic Virus (CaMV) was used as the promoter. The 3 'end of the octopine synthase gene served as the termination signal.

Transport into chloroplasts was achieved by fusion of the SAT mutant with the plastid import peptide of plastid transketolase from tobacco in pBinAr-TkTp. Localization in cytosol was done with pBinAR, leaving out the section for the import peptide.

The cloning was carried out in the BamHI or Sall restriction sites of the vectors pBIN-AR and pBINAr-TkTp. The cDNA of the SAT mutant was amplified with the oligonucleotide primers SAT208 and SAT209 with 5 'additional BamHI and Sall restriction cleavage sites by standard PCR. Example of standard PCR: reaction volume 50 μl with 20 pmol of each primer, 1-10 ng plasmid, buffer from the manufacturer, 1 U Taq polymerase (Promega). Sequence: 5 minutes at 94 ° C, then 30 cycles of 30 seconds at 94 ° C, 60 seconds at 55 ° C, 30 seconds at 72 ° C, followed by 10 minutes at 72 ° C.

SAT208: 5'-GGATCC CAT GAA CTA CTT CCGTTATC-3 'SAT209: 5'-GTC GAC TCAAAT TAG ATAATC CGA C-3'

Determination of the thiol and amino acid content of transgenic plants

Cysteine, glutathione and methionine levels were determined in leaves of at least three independent first and second generation transgenic tobacco plants after transformation. In comparison to non-transformed control plants, up to 8x more cysteine and 2x more glutathione were found in cytosolic dominant-negative overexpression. With corresponding plastid overexpression of the SAT mutant, up to 25x more cysteine and 8x more glutathione were measured. With plastid overexpression, the methionine content increases up to 2x.

The extraction, separation and quantification of cysteine and glutathione was carried out as described (Hell and Bergmann (1990) Planta 180, 603-612). The determination of methionine was carried out according to a commercial method (Waters, Eschborn) as described (Giordano et al. (2000) Plant Physiol. 124, 857-864).

The results described above clearly showed that the Arabidopsis thaliana SAT mutant with an amino acid exchange in position 309 no longer has any enzymatic SAT activity, but is nevertheless able to form a complex, ie to interact with OAS-TL. The expression of this mutant led to a surprising increase in the content of sulfur compounds, especially cysteine, glutathione and methionine, in transgenic plants.

pictures

Figure 1 shows an amino acid alignment of various serine acetyltransferases.

Figure 2 shows the influence of overexpression of the SAT A mutant H309A on the formation of cysteine.

Claims

PATENT CLAIMS

1. A method for increasing the content of sulfur-containing compounds such as cysteine, glutathione and / or methionine in plants by transfer and expression of DNA sequences which code for a serine acetyltransferase in plants, characterized in that the serine acetyltransferase is enzymatically inactive.

2. The method according to claim 1, wherein the serine acetyltransferase for

Interaction with O-acetylserine (thiol) lyase is capable.

3. The method of claim 1 or 2, wherein the serine acetyltransferase is enzymatically inactive due to a mutation within the amino acid sequence motif G K X X G D R H P K I G D (X stands for any amino acid).

4. The method according to claim 3, wherein the mutation relates to the core motif D R H.

5. The method of claim 3 or 4, wherein the mutation affects the histidine within the motif.

6. The method of claim 1 or 2, wherein the serine acetyltransferase is enzymatically inactive due to binding and inhibition by a ligand or a protein or an antibody.

7. Recombinant nucleic acid molecule containing a promoter active in plant cells in operative linkage with a DNA sequence which codes for an enzymatically inactive serine acetyltransferase, the enzymatic Inactivity is caused by a mutation in the amino acid sequence motif GKXXGDRHPKIGD (X stands for any amino acid).

8. Recombinant nucleic acid molecule according to claim 7, wherein the mutation lies in the core motif D R H.

9. Recombinant nucleic acid molecule according to claim 7 or 8, wherein the mutation relates to the histidine.

10. Enzymatically inactive serine acetyltransferase, which interacts with O-

Acetylserine (thiol) lyase is capable.

11. Serine acetyltransferase according to claim 10, which is enzymatically inactive due to a mutation within the amino acid sequence motif G K X X G D R H P K I G D (X stands for any amino acid).

12. Serine acetyltransferase according to claim 11, wherein the mutation relates to the core motif D R H.

13. Serine acetyltransferase according to claim 11 or 12, wherein the mutation affects the histidine within the motif.

14. A method for producing plants with a content of sulfur-containing compounds such as cysteine, glutathione and / or methionine which is higher than that of wild type plants, comprising the following steps:

- The transfer of a recombinant nucleic acid molecule or vector containing a DNA sequence coding for an enzymatically inactive serine acetyltransferase, which is able to interact with O-acetylserine (thiol) lyase, on plant cells; - the regeneration of plants from the transformed plant cells.

15. Transgenic plants, produced in a process according to one of claims 1 to 6 or 14 or containing a recombinant nucleic acid molecule according to one of claims 7 to 9, and parts of these plants, transgenic crop products and transgenic propagation material of these plants such as

Plant cells, protoplasts, calli, seeds, tubers, cuttings and the transgenic progeny of these plants.

16. Use of the method according to one of claims 1 to 6 or 14 to increase the stress resistance in the transgenic plants.