AU1783000A - Method for increasing the content in sulphur compounds and particularly in cysteine, methionine and glutathione in plants and plants obtained - Google Patents

Description

WO 00/36127 PCT/FR99/03179 1 Method for increasing the content of sulphur compounds and in particular of cysteine, methionine and glutathione in plants and plants obtained Methionine is the first limiting essential 5 amino acid in plants, in particular the leguminous plants which are one of the basic elements of the animal diet. Cysteine, another sulphur-containing amino acid, is not an essential amino acid, but can be taken to be a limiting element for animal nutrition since 10 cysteine is derived, in animals, from methionine. In maize, the sulphur-containing amino acids are also limiting amino acids after lysine and tryptophan. The reason for this is that the major storage proteins of the seeds of these plants are lacking in these amino 15 acids. The overproduction of methionine and cysteine in the seeds of leguminous plants (soybean, lucerne, pea, etc.) and of maize will thus have a considerable impact on the nutritional quality of these seeds. So far, the increase in the nutritional 20 quality of foods derived from the seeds of leguminous plants has been obtained by supplementation with chemically synthesized free methionine. For example, the average contents of methionine + cysteine in soybean and pea are of the order of 20 mg per g of 25 protein. This content must be increased to a value of the order of 25 mg cysteine + methionine/g of protein to cover the dietary needs of a human adult, and to a 2 value of the order of 48 mg of cysteine + methionine/g of protein to cover those of pigs (De Lumen, B.O., Food Technology (1997) 51, 67-70). The techniques for characterizing proteins 5 enriched in sulphur-containing amino acids and the preparation of transgenic plants allowing the expression of such proteins, so as to increase the sulphur-containing amino acid content of these plants and thus their nutritive value for the animal diet, and 10 thus to diminish the amount of synthesized methionine supplied, are now well known and described in the literature ([1] Korit, A.A. et al., Eur. J. Biochem (1991) 195, 329-334; WO 98/20133; WO 97/41239; WO 95/31554; WO 94/20828; WO 92/14822). 15 The enrichment in proteins with a high sulphur-containing amino acid content by such an approach remains, however, limited by the capacity of plant cells and of plants to produce the said sulphur containing amino acids required for the synthesis of 20 the protein. The reason for this is that plants overexpressing a protein rich in methionine and cysteine in their seed, such as for example lupins expressing 8S albumin, contain a level of free methionine and cysteine, and also of glutathione, which 25 is lower than that of control plants ([2] Tabe, L. & Droux, M., 4th Workshop on Sulphur Metabolism, in press).

3 In the same way, peptides rich in sulphur containing amino acids and having antifungal or antibacterial activity have been identified (WO 97/30082, WO 99/02717, WO 99/09184, WO 92/24594, 5 WO 99/53053). The expression of these peptides in the plants makes it possible to increase the capacity of the said plants to resist certain fungal or bacterial attacks. Here again, the production of such peptides in the plants remains limited by the capacity of plant 10 cells and plants to produce the sulphur-containing amino acids required for the synthesis of these peptides. The reason for this is that the expression of these peptides in the plant cell occurs to the detriment of the stock of glutathione, which is taken 15 to be a reservoir for cysteine. It has been observed that the limiting parameter of such an approach is indeed linked to this capacity to produce methionine or cysteine. It is therefore important to be able to modify in the plants 20 this capacity to produce methionine and cysteine in sufficient quantities to allow the production of heterologous proteins with a high sulphur-containing amino acid content, that is to say to use a molecular strategy intended to increase the levels of cysteine 25 and methionine in plants, and more particularly, crop plants of agronomical interest.

4 In plants, methionine biosynthesis is carried out from cysteine, this same cysteine being involved in the synthesis of glutathione. Glutathione is a form of storage of reduced 5 sulphur and represents 60 to 70% of the organic sulphur in the cell. Glutathione plays an important role for plants in the resistance to oxidative stress and in the elimination of toxic compounds. It thus participates in the elimination of xenobiotic compounds: heavy metals 10 (for example) via the formation of phytochelatins and metallothionines; herbicides, via glutathione S-transferase activity; which are toxic to the plant, and in the plant's defence mechanisms against micro organisms. By increasing a plant's cysteine content, 15 and consequently its glutathione content, it is thus possible to modulate the plant's response to the different stresses mentioned above. There are therefore two distinct metabolic pathways starting from cysteine, one for the 20 preparation of methionine, the other for the preparation of glutathione (Figure 1) and for which the different enzymes involved are recalled below. The SAT (El) and OASTL (E2) activities are at a metabolic crossroads between the assimilation of organic nitrogen 25 and carbon (serine) and of inorganic sulphur (reduced sulphur from the sequence of assimilation and reduction of sulphate, shaded box). The cysteine is then incorporated into proteins, but also participates in 5 the synthesis of glutathione and methionine. The synthesis of the carbon backbone (0-phosphohomoserine) of this latter amino acid, is derived from aspartate. Aspartate is also the precursor for lysine, threonine 5 and isoleucine synthesis. Moreover, the presence of a potentially limiting step for the synthesis of methionine by transcriptional regulation of CGS (cystathionine y-synthase) is indicated in the diagram ([3] Giovanelli J. in Sulphur Nutrition and Sulphur 10 Assimilation in Higher Plants, (1990) pp. 33-48; [4] Chiba Y. et al. (1999), Science, 286, 1371-1374). Methionine is the precursor of SAM (S-adenosylmethionine) which is involved in most methylation reactions, and of SMM (S-methylmethionine) 15 taken to be a transport form and a storage form of methionine ([3]). In plants the final steps of cysteine synthesis involve the two enzymes below: El) Serine acetyltransferase (EC 2.3.1.30)(SAT): 20 Serine + acetyl-coenzyme A 4 O-acetylserine + coenzyme A E2) O-acetylserine (thiol) lyase (EC 4.2.99.8)(OASTL): 0-acetylserine + sulphide + cysteine + acetate The synthesis of methionine from cysteine 25 involves, successively, the three enzymes below: E3) cystathionine y-synthase (EC 4.2.99.9)(CGS): 0-phosphohomoserine + cysteine -4 cystathionine + Pi Pi signifies inorganic phosphate.

6 E4) cystathionine S-1yase (EC 4.4.1.8)(CBL): cystathionine + H 2 0 4 homocysteine + pyruvate + NH 4 + E5) methionine synthase (EC 2.1.1.14)(Ms): homocysteine + 5-methyltetrahydrofolate 4 methionine + 5 tetrahydrofolate As for the synthesis of glutathione from cysteine, it involves, successively, the two enzymes below: E6) y-glutamylcysteine synthetase (EC 6.3.2.2) 10 glutamate + L-cysteine + ATP + y-glutamylcysteine + ADP + Pi E7) glutathione synthetase (EC 6.3.2.3) y-glutamylcysteine + glycine + ATP 4 glutathione + ADP + Pi 15 All these enzymes have been characterized and cloned in plants ([5] Lunn, J.E. et al., Plant Physiol. (1990) 94, 1345-1352; [6] Rolland, N. et al., Plant Physiol. (1992) 98, 927-935; [7] Droux, M. et al., Arch. Biochem. Biophys. (1992) 295, 379-390; 20 [8] Rolland, N. et al., Arch. Biochem (1993) 300, 213 222; [9] Ruffet, M.L. et al., Plant Physiol. (1994) 104, 597-604; [10] Ravanel, S. et al., Arch. Biochem. Biophys. (1995) 316, 572-5584; [11] Droux, M. et al., Arch. Biochem. Biophys. (1995) 31, 585-595; 25 [12] Ruffet, M.L. et al., Eur. J. Biochem. (1995) 227, 500-509; [13] Ravanel, S. et al., Biochem. J. (1996) 320, 383-392; [14] Ravanel, S. et al., Plant Mol. Biol. (1996) 29, 875-882; [15] Rolland, N. et al., Eur. J.

7 Biochem. (1996) 236, 272-282; [161 Ravanel, S. et al., Biochem. J. (1998) 331, 639-648; [17] Droux, M. et al., Eur. J. Biochem. (1998) 255, 235-245; [18] May, M.J., Leaver, C.J., Proc. Natl. Acad. Sci. USA (1994) 91, 5 10059-10063; [19] Ullmann, P. et al., Eur. J. Biochem. (1996) 236, 662-669; [20] Eichel, J. et al., Eur. J. Biochem. (1995) 230, 1053-1058). It is known that for cysteine synthesis, the El and E2 enzymes are present in the three compartments 10 of the plant cell, that is to say, the plasts, the cytosol and the mitochondria (5-6, 9, 12). These three El enzymes are named SAT2 and SAT4 for the (putative) chloroplast enzyme, and SAT1 for the mitochondrial enzyme, and SAT3 and SAT3' (SAT52) for the cytoplasmic 15 enzyme. These localization attributions are based on sequence analysis. For the methionine synthesis enzymes, the situation is different since the E3 and E4 enzymes are exclusively localized in the plasts (10-11, 13-14, 16), 20 while the terminal E5 enzyme is in the cytosol (20). As for the enzymes associated with the glutathione biosynthetic pathway, they are localized both in the chloroplast and in the cytosol ([21] Hell, R. and Bergmann, L., Planta (1990) 180, 603-612). 25 The E3 enzyme, of the methionine synthetic pathway, has a Km (substrate concentration giving the half-maximal rate) of the order of 200 AM to 500 gM for 8 cysteine (10, 16, [22] Kreft, B-D. et al., Plant Physiol. (1994) 104, 1215-1220). The E6 enzyme, of the glutathione synthetic pathway, also has a high Km for cysteine, of the order 5 of 200 gM [21]. It has now been observed the chloroplast serine acetyltransferase enzyme (Figure 2) and to a lesser degree the mitochondrial SAT are inhibited by cysteine, in contrast to the cytoplasmic enzyme (Figure 10 2), this inhibition constituting the essential limiting factor in the synthesis of cysteine in plant cells and being downstream of the methionine and glutathione. The present invention thus consists in increasing the level of cysteine and methionine 15 synthesized in the cellular compartments of plant cells, and in particular in the chloroplast compartment. Increasing the level of cysteine, the sulphur-containing precursor of glutathione and of methionine and its derivatives, advantageously makes it 20 possible to increase the level of methionine and/or of glutathione in the plant cells and plants, and subsequently to improve the production of proteins, natural or heterologous, enriched in sulphur-containing amino acids in the plant cells and plants, and 25 similarly the tolerance of the plants to different forms of glutathione-regulated stress.

9 This increase according to the invention is obtained by overexpressing a serine acetyltransferase (SAT) in the plant cells and plants. The present invention thus relates to a 5 method for increasing the production of cysteine, glutathione, methionine and sulphur-containing derivatives thereof, by plant cells and plants, the said method consisting in overexpressing an SAT in the plant cells and in plants containing the said plant 10 cells. The overexpressed SAT can consist of any SAT, whether of plant origin, in particular SAT2 or SAT4, SAT1, SAT3, SAT3' (SAT52), or of any other origin, in particular bacterial, in a native or mutant form or 15 deleted of a fragment, and functional in the synthesis of O-acetylserine. In particular, it can be a cysteine-sensitive SAT, such as for example a plant SAT, for example a chloroplast or mitochondrial SAT (SAT2, SAT4, SAT1), or 20 a native SAT of bacterial origin ([22] Nakamori et al., 1998, Appl. Environ, Microbiol., 64, 1607-1611; [23] Takagi H. et al., 1999, Febs Lett. 452, 323-327; [24] Mino K. et al., 1999, Biosci. Biotechnol. Biochem., 63, 168-179). 25 It can also be a cysteine-insensitive SAT, such as, for example, a plant SAT, for example a cytoplasmic SAT (SAT3), or a mutant SAT of bacterial origin, made insensitive to cysteine by mutagenesis 10 ([22] and [23], whose contents are incorporated here by reference), or any mutant plant SAT which is functional in the synthesis of O-acetylserine (the carbon containing precursor for cysteine synthesis). 5 According to a specific embodiment of the invention, the SAT is an Arabidopsis thaliana SAT [12]. According to a first embodiment of the invention, the SAT is overexpressed in the cytoplasm of the plant cells. The SAT is either a plant cytoplasmic 10 SAT, in particular the SAT3 (L34076) or SAT3' or SAT52 (U30298), represented by the SEQ ID NO 1 or the SEQ ID NO 2, respectively, or an SAT of bacterial origin as defined above. The SAT overexpressed in the cytoplasm can also be a noncytoplasmic plant SAT, for example a 15 chloroplast or mitochondrial SAT. These noncytoplasmic plant SATs, naturally, are expressed in the cytoplasm in the form of a precursor protein comprising a signal for addressing to the cellular compartment, other than the cytoplasm, into which the mature functional SAT is 20 released. In order to overexpress these mature functional SATs in the cytoplasm, their addressing signal is removed. In this case, the SAT protein overexpressed in the cytoplasm is a noncytoplasmic plant SAT, with its signal(s) for addressing to 25 cellular compartments, other than the cytoplasm, removed.

11 According to a specific embodiment of the invention, the noncytoplasmic SAT with its addressing signal removed is SAT1' represented by SEQ ID NO 3. According to a second embodiment of the 5 invention, the SAT is overexpressed in the mitochondria. The protein is advantageously expressed in the cytoplasm in the form of a signal peptide/SAT fusion protein, the mature functional SAT being released inside the mitochondria. Advantageously, the 10 mitochondrial addressing signal peptide is made up of at least one mitochondrial addressing signal peptide from a plant protein which is located in mitochondria, such as the tobacco ATPase -F1 subunit signal peptide [[25] Hemon P. et al. 1990, Plant Mol. Biol. 15, 895 15 904], or the SATl signal peptide represented by amino acids 1 to 63 in SEQ ID NO 4. According to a specific embodiment of the invention, the mitochondrial SAT is SATl (U22964) represented by SEQ ID NO 4. 20 According to a third embodiment of the invention, the SAT is overexpressed in the chloroplasts of the plant cells. The SAT will be expressed in the chloroplasts by any appropriate means, in particular by any means 25 known to persons skilled in the art and widely described in the prior art. According to a specific embodiment of the invention, the SAT is overexpressed in the chloroplasts 12 by integrating into the chloroplast DNA a chimeric gene comprising a DNA sequence encoding the said SAT, under the control of 5' and 3' regulatory elements that function in the chloroplasts. The techniques for 5 insertion of genes into chloroplasts, such as the regulatory elements appropriate for the expression of the said genes in chloroplasts, are well known to persons skilled in the art and in particular are described in the following patents and patent 10 applications: US 5,693,507, US 5,451,513 and WO 97/32977. According to another specific embodiment of the invention, the SAT is overexpressed in the cytoplasm in the form of a transit peptide/SAT fusion 15 protein, the function of the transit peptide being to address the SAT to which it is fused to the chloroplasts, the mature functional SAT being released inside the chloroplasts after cleavage at the chloroplast membrane. 20 In this case, the SAT can be a chloroplast SAT of plant origin, such as SAT2 or SAT4, represented by SEQ ID NO 5 or 6, respectively. The SAT can also be a cytoplasmic SAT of plant origin or an SAT of bacterial origin as defined 25 above. The cytoplasmic SATs are understood to include also noncytoplasmic SATs from which have been removed their signal for addressing to a compartment other than the cytoplasm, as defined above.

13 The transit peptides, their structures, their methods of functioning and their use in the construction of chimeric genes for addressing a heterologous protein into chloroplasts, as well as 5 chimeric transit peptides comprising several transit peptides, are well known to persons skilled in the art and widely described in the literature. In particular, the following patent applications are mentioned: EP 189 707, EP 218 571 and EP 508 909, and the 10 references cited in these patent applications, whose contents are incorporated here by reference. In the fusion protein according to the invention, the SAT can be homologous or heterologous to the transit peptide. In the first case, the fusion 15 protein is the SAT2 or the SAT4 protein expressed naturally in the chloroplasts of plant cells. In the second case, the transit peptide can be a transit peptide from an SAT2, represented by amino acids 1 to 32 of SEQ ID 5, or the transit peptide from an SAT4, 20 represented by amino acids 1 to 30 of SEQ ID NO 6, or alternatively a transit peptide from another protein, which is located in plastids, in particular the transit peptides defined below. Protein which is located in plastids is understood to mean a protein expressed in 25 the cytoplasm of plant cells in the form of a transit peptide/protein fusion protein, the mature protein being localized in the chloroplast after cleavage of the transit peptide.

14 A plant EPSPS transit peptide is, in particular, described in Patent Application EP 218,571, while a plant RuBisCO ssu transit peptide is described in Patent Application EP 189,707. 5 According to another embodiment of the invention, the transit peptide also comprises, between the C-terminal region of the transit peptide and the N-terminal region of the SAT a portion of sequence from the mature N-terminal region of a protein which is 10 located in plastids, this portion of sequence generally comprising less than 40 amino acids from the N-terminal region of the mature protein, preferably less than 30 amino acids, more preferably between 15 and 25 amino acids. Such a transit peptide comprising a transit 15 peptide fused to a part of the N-terminal region of a protein which is located in plastids is, in particular, described in Patent Application EP 189,707, more particularly for the transit peptide and the N-terminal region of plant RuBisCO ssu. 20 According to another embodiment of the invention, the transit peptide also comprises, between the C-terminal region of the N-terminal region of the mature protein and the N-terminal region of the SAT, a second transit peptide from a plant protein which is 25 located in plastids. Preferably, this chimeric transit peptide comprising a combination of several transit peptides, is an optimized transit peptide (OTP) made by fusing a first transit peptide with a portion of 15 sequence from the mature N-terminal region of a protein which is located in plastids, which is fused with a second transit peptide. Such an optimized transit peptide is described in Patent Application EP 508,909, 5 more particularly, the optimized transit peptide comprising the sunflower RuBisCO ssu transit peptide fused to a peptide made of the 22 N-terminal amino acids of the mature maize RuBisCO ssu, fused to the maize RuBisCO ssu transit peptide. 10 The present invention also relates to a transit peptide/SAT fusion protein in which the SAT defined above is heterologous to the transit peptide and in which the transit peptide is made of at least one transit peptide from a natural plant protein which 15 is located in plastids, as defined above. The present invention also relates to a nucleic acid sequence encoding a transit peptide/SAT fusion protein, described above. According to the present invention, "nucleic acid sequence" is 20 understood to mean a nucleotide sequence which can be of DNA or RNA type, preferably of DNA type, in particular double-stranded, whether of natural or synthetic origin, in particular a DNA sequence in which the codons encoding the fusion protein according to the 25 invention have been optimized according to the host organism in which it will be expressed, these optimization methods being well known to persons skilled in the art.

16 A subject of the invention is also the use of a nucleic acid sequence encoding an SAT according to the invention defined above, in particular for chloroplast, mitochondrial or cytoplasmic addressing, 5 in a method for transforming plants, as a coding sequence allowing the modification of the cysteine, methionine, methionine derivatives, and glutathione contents of the transformed plants. This sequence can of course also be used in combination with other marker 10 gene(s) and/or coding sequence(s) for one or more other agronomic properties. The present invention also relates to a chimeric gene (or expression cassette) comprising a coding sequence as well as heterologous 5' and 3' 15 regulatory elements capable of functioning in a host organism, in particular plant cells or plants, the coding sequence comprising at least one nucleic acid sequence encoding an SAT as defined above. Host organism is understood to mean any 20 monocellular or pluricellular higher or lower organism, into which the chimeric gene according to the invention can be introduced. They are in particular bacteria, for example E. coli, yeasts, in particular of the genera Saccharomyces, Kluyveromyces or Pichia, fungi, in 25 particular Aspergillus, a baculovirus, or preferably plant cells and plants. "Plant cell" is understood to mean according to the invention any cell derived from a plant and 17 capable of constituting undifferentiated tissues such as calli, differentiated tissues such as embryos, plant portions, plants or seeds. "Plant" is understood to mean according to 5 the invention any differentiated multicellular organism capable of photosynthesis, in particular monocotyledonous or dicotyledonous plants, more particularly crop plants intended or not as animal feed or for human consumption, such as maize, wheat, rape, 10 soybean, rice, sugar cane, beet, tobacco, cotton and the like. The regulatory elements required for the expression of the a nucleic acid sequence encoding a fusion protein according to the invention are well 15 known to persons skilled in the art according to the host organism. They comprise, in particular, promoter sequences, transcription activators, termination sequences including start and stop codons. The means and methods of identifying and selecting the regulatory 20 elements are well known to persons skilled in the art and widely described in the literature. The invention relates more particularly to the transformation of plants. Promoter regulatory sequences which can be used in plants, are any promoter 25 sequence of a gene which is naturally expressed in plants, in particular a promoter which is expressed in particular in the leaves of plants such as, for example, so-called constitutive promoters of bacterial, 18 viral or plant origin, or alternatively so-called light-dependent promoters such as that of a plant ribulose-biscarboxylase/oxygenase (RuBisCO) small subunit gene or any suitable known promoter that can be 5 used. Among promoters of plant origin which can be mentioned are the histone promoters as described in Application EP 0,507,698, or the rice actin promoter (US 5,641,876). Among promoters of plant virus genes which can be mentioned are that of the cauliflower 10 mosaic (CAMV 19S or 35S), or the circovirus promoter (AU 689 311). It is also possible to use a promoter regulatory sequence which is specific for regions or tissues specific to plants, and more particularly seed 15 specific promoters ([26] Datla, R. et al., Biotechnology Ann. Rev. (1997) 3, 269-296), in particular the napin (EP 255,378), phaseolin, glutenin, zein, helianthinin (WO 92/17580), albumin (WO 98/45460), oelosin (WO 98/45461), ATS1 or ATS3 20 (WO 99/20275) promoters. According to the invention, it is also possible to use, in combination with the regulatory promoter sequence, other regulatory sequences which are situated between the promoter and the coding sequence, 25 such as transcription enhancers, such as, for example the translational enhancer of tobacco mosaic virus (TMV) described in Application WO 87/07644, or of 19 tobacco etch virus (TEV) described by Carrington & Freed. Regulatory termination or polyadenylation sequences which can be used, are any corresponding 5 sequence of bacterial origin, such as for example the nos terminator of Agrobacterium tumefaciens, or alternatively of plant origin, such as for example a histone terminator as described in Application EP 0,633,317. 10 The present invention also relates to a cloning and/or expression vector for the transformation of a host organism containing at least one chimeric gene as defined above. This vector comprises, besides the chimeric gene above, at least one origin of 15 replication. This vector can be a plasmid, a cosmid, a bacteriophage or a virus, which has been transformed by introducing a chimeric according to the invention. Such transformation vectors, according to the host organism to be transformed, are well known to persons skilled in 20 the art and widely described in the literature. For the transformation of plant cells or plants, a virus, moreover containing its own elements of replication and expression, can, in particular, be used to transform developed plants. Preferably, the transformation vector 25 of plant cells or plants according to the invention is a plasmid. For the transformation of host organisms, the chimeric gene according to the invention can be used in 20 combination with a selection marker gene, either in the same vector, the two genes being combined in a convergent, divergent or colinear manner, or alternatively in two vectors used simultaneously for 5 transforming the host organism. Such marker genes and their use for transforming host organisms are well known to persons skilled in the art and widely described in the literature. Among genes encoding selection markers which 10 can be mentioned are antibiotic-resistance genes, genes which impart tolerance to herbicides (bialaphos, glyphosate or isoxazoles), genes encoding easily identifiable enzymes such as the GUS enzyme (or GFP, "Green Fluorescent, Protein"), or genes encoding 15 pigments or enzymes which regulate the production of pigments in the transformed cells. Such selection marker genes are in particular described in Patent Applications EP 242 236, EP 242 246, GB 2 197 653, WO 91/02071, WO 95/06128, WO 96/38567 or WO 97/04103. 20 The subject of the invention is also a method for transforming host organisms, in particular plant cells, by integration of at least one nucleic acid sequence or one chimeric gene as defined above, which transformation may be obtained by any known appropriate 25 means, widely described in the specialist literature and in particular the references cited in the present application, more particularly by the vector according to the invention.

21 One series of methods consists in bombarding cells, protoplasts or tissues with particles to which the DNA sequences are attached. Another series of methods consists in using, as a means of transferring 5 into the plant, a chimeric gene inserted into an Agrobacterium tumefaciens Ti plasmid or an Agrobacterium rhizogenes Ri plasmid. Other methods can be used, such as microinjection or electroporation, or alternatively direct or PEG precipitation. Persons 10 skilled in the art will choose the appropriate method according to the nature of the host organism, in particular of the plant cell or of the plant. The subject of the present invention is also the host organisms, in particular plant cells or 15 plants, which are transformed and which contain a chimeric gene defined above. The subject of the present invention is also the plants containing transformed cells, in particular the plants regenerated from the transformed cells. The 20 regeneration is obtained by any appropriate method which depends on the nature of the species, as for example described in the above references. Patents and patent applications which are mentioned for the methods of transforming plant cells and of regenerating plants 25 are, in particular, the following: US 4,459,355, US 4,536,475, US 5,464,763, US 5,177,010, US 5,187,073, EP 267,159, EP 604,662, EP 672,752, US 4,945,050, US 5,036,006, US 5,100,792, US 5,371,014, US 5,478,744, 22 US 5, 179,022, US 5,565,346, US 5,484,956, US 5,508,468, US 5,538,877, US 5,554,798, US 5,489,520, US 5,510,318, US 5,204,253, US 5,405,765, EP 442,174, EP 486,233, EP 486,234, EP 539,563, EP 674,725, 5 WO 91/02071 and WO 95/06128. The subject of the present invention is also the transformed plants derived from the cultivation and/or the crossing of the above regenerated plants, as well as the seeds of the transformed plants. 10 The transformed plants which can be obtained according to the invention can be of monocotyledonous type, such as for example cereals, sugar cane, rice and maize, or of dicotyledonous type, such as for example tobacco, soybean, rape, cotton, beet, clover, etc. 15 The plants transformed according to the invention can contain other genes of interest, which confer novel agronomic properties on the plants. Among genes conferring novel agronomic properties on the transformed plants which can be mentioned are genes 20 conferring tolerance to certain herbicides, those conferring tolerance to certain insects, and those conferring tolerance to certain diseases. Such genes are in particular described in Patent Applications WO 91/02071 and WO 95/06128. Mention may also be made 25 of genes which modify the composition of the modified plants, in particular the content and quality of certain essential fatty acids (EP 666,918), or alternatively the content and quality of proteins, in 23 particular in the leaves and/or seeds of the said plants. In particular, genes encoding proteins enriched in sulphur-containing amino acids are cited([1]; WO 98/20133; WO 97/41239; WO 95/31554; WO 94/20828; 5 WO 92/14822; US 5,939,599, US 5,912,424). The function of these proteins enriched in sulphur-containing amino acids is also to trap and store surplus cysteine and/or methionine, making it possible to avoid any problems of toxicity linked to an overproduction of these sulphur 10 containing amino acids, by trapping them. Mention may also be made of genes encoding peptides rich in sulphur-containing amino acids and more particularly rich in cysteine, the said peptides also having antibacterial and/or antifungal activity. 15 More particularly, plant defensins are mentioned, as well as lytic peptides of any origin, and more particularly the following lytic peptides: androctonin (WO 97/30082 and WO 99/09189), drosamicin (WO 99/02717), thanatin (WO 99/24594) or heliomicin 20 (WO 99/53053). These other genes of interest can be combined with the chimeric gene according to the invention either by conventional crossing of two plants each containing one of the genes (the first being the 25 chimeric gene according to the invention and the second being the gene encoding the protein of interest), or by transforming the plant cells of a plant containing the 24 gene encoding the protein of interest, with the chimeric gene according to the invention. The following examples illustrate the invention, without, however, looking to limit its 5 scope. All of the methods or operations described below in these examples are given by way of examples and correspond to a choice made from the different methods available to arrive at the same result. This 10 choice has no bearing on the quality of the result and consequently, any adapted method can be used by persons skilled in the art to arrive at the same result. Most of the methods for engineering DNA fragments are described in "Current Protocols in Molecular Biology" 15 Volumes 1 and 2, Ausubel F.M. et al, published by Greene Publishing Associates and Wiley Interscience (1989) or in Molecular Cloning, T. Maniatis, E.F. Fritsch, J. Sambrook, 1982. The contents of all the references cited in 20 the above description and in the following examples are incorporated into the text of the present patent application by reference. Example 1. Demonstration of the inhibition of 25 chloroplast serine acetyltransferase from pea (Pisum sativum) leaves by cysteine The three subcellular compartments corresponding to the cytosol (preparation from a 25 subcellular fractionation of pea protoplasts, [12]), to mitochondria and to chloroplasts are prepared from pea leaves [121. The soluble proteins are extracted therefrom and the serine acetyltransferase activity 5 present in each of the compartments is measured by means of a described technique [12, 17]. Description of the assay method: The serine acetyltransferase activity is 10 measured by high performance liquid chromatography (HPLC), by assaying the O-acetylserine formed during the course of the reaction (reaction 1), after derivatization with orthophthalaldehyde (OPA). A defined quantity of the protein extract, corresponding 15 to the cytosol and to the different soluble fractions of chloroplasts (stroma) and of mitochondria (matrix), is desalted on a PD10 column (Pharmacia) pre equilibrated in a buffer containing 50 mM Hepes-HCl, pH 7.5 and 1 mM EDTA. The enzyme reaction is carried 20 out in the presence of 50 mM Hepes-HCl, pH 7.5, 1 mM dithiothreitol, 10 mM L-serine, 2.5 mM acetyl-CoA, in a 100 gl reaction volume, at 25oC. After 10 to 15 minutes' incubation, the reaction is stopped by addition of 50 gl of 20% (W/V) trichloroacetic acid. 25 The proteins thus precipitated are then eliminated by centrifugation for 2 min at 15,000 g. The supernatant, which contains the reaction product (OAS), is mixed with 500 yl of a derivatization solution (54 mg of OPA 26 dissolved in 1 ml of absolute ethanol, 9 ml of a 400 mM solution of borate-NaOH, pH 9.5, and 0.2 ml of 14 M $-mercaptoethanol) and incubated for 2 min. A fraction of this mixture (20 4l) is injected onto a reverse 5 phase column (3.9 X 150 mm, AccQ Tag C 18 column, Waters) which is connected to an HPLC system. The buffers used to elute the compounds derivatized by OPA are: Buffer A, 85 mM sodium acetate, pH 4.5 and 6% (V/V) acetonitrile, pH 4.5; Buffer B, 60% (V/V) acetonitrile 10 in water. The O-acetylserine, which has been derived by OPA, is eluted with a continuous linear gradient of buffer B in buffer A, of 25 to 70% (V/V), and is detected by fluorescence at 455 nm (excitation at 340 nm). The retention time of O-acetylserine, under 15 our conditions, is of the order of 6.2 min., and the amount of product which is formed in the enzyme assays is quantified from a standard curve which is obtained for O-acetylserine. The enzyme assays were optimized in order to respect the optimum pH of the reaction, the 20 linearity with time, and in order to operate under saturating conditions of substrates. Effect of cysteine on serine acetyltransferase activity of pea leaves 25 Incubation (2 min) is carried out in the presence of protein extract (cytosol, matrix, and stroma), and in the presence of increasing concentrations of L-cysteine (from 0 to 1 mM), before 27 adding saturating concentrations of the serine acetyltransferase substrates, L-serine (10 mM) and acetyl-CoA (2.5 mM). The enzyme reaction and assay of residual 0-acetylserine in the supernatant are carried 5 out as described above. The result of these experiments is represented in the graph of Figure 2, in the annex. If free cysteine (from 0 to 1 mM, Figure 2) is added to the different assays, a very strong inhibition of chloroplast serine acetyltransferase 10 activity is observed (inhibition constant of the order of 30 gM). Mitochondrial serine acetyltransferase activity is inhibited, but at higher concentrations of cysteine (inhibition constant of the order of 300 gM). On the other hand, cytosolic serine acetyltransferase 15 activity is insensitive to inhibition by cysteine up to concentrations of the order of 1 mm cysteine (Figure 2). This result proves, therefore, that only chloroplast serine acetyltransferase activity, thus the enzyme associated with the sulphate assimilation 20 pathway, is inhibited by the final product, L-cysteine.

28 Table I: Determination of the specific activities and

IC

50 values of cysteine for each of the serine acetyltransferase isoforms. Serine acetyltransferase (Pisum sativum) Specific activity IC 50 L-cysteine nmol OAS-min~1-mg'1 AM Stroma 0.93 ± 0.2 33.4 8 Matrix 10 ±2 283 50 Cytosol 0.83 ± 0.3 no inhibition 5 The concentration of L-cysteine which makes it possible to obtain 50% inhibition (IC 5 0 ) under standard reaction conditions, and which is calculated for different enzyme preparations, is represented in 10 Table I. Determination of the serine acetyltransferase enzyme activity and of the IC 50 is carried out for 9 different experiments (on stroma), and for 3 experiments for the cytosolic extracts and 3 for the mitochondrial extracts. Similarly, activity of 15 chloroplast serine acetyltransferase from spinach leaves is cysteine-sensitive. Conversely, in Arabidopsis thaliana, only the activity of the isoform associated with the cytosolic compartment seems to be controlled by the level of cysteine ([27] Noji M. et 20 al. 1998, J. Biol. Chem. 273, 32739-32745; [28] Inoue K. et al. 1999, Eur. J. Biochem. 266, 220-227). For 29 these authors, the activity associated with the chloroplast compartment is cysteine-insensitive. It would seem, therefore, that inhibition of the chloroplast serine acetyltransferase activity by 5 cysteine is a plant-specific phenomenon, but, in particular, is very pronounced in leguminous plants, such as pea. Study of the mode of inhibition of serine 10 acetyltransferase activity by cysteine The enzyme reaction rate was determined for fixed concentrations of cysteine (0 gM; 10 gM; 20 gM; 40 gMm 60 gM and 100 gM), by varying either the L-serine concentration or the acetyl-CoA concentration, 15 for saturating concentrations of the second co substrate. For each of the kinetics obtained, the affinity of the enzyme for these substrates does not seem to be affected, but, on the other hand, the maximum reaction rate is modified. The more the 20 concentration of L-cysteine increases, the more the rate of O-acetylserine synthesis decreases. For each of the conditions analysed, the inhibition constant Ki was estimated to be of the order of 30 (±2.2) AM (variable substrate: serine), and 22 (±2) gM (variable substrate: 25 acetyl-CoA). We were able to show that cysteine is a non-competitive type of inhibitor of serine acetyltransferase activity and that, moreover, it is an allosteric type inhibitor (Hill constant of the order 30 of 1.6±0.3 gM), using conventional kinetics equations ([29] Segel, I.H. (1995), John Wiley and Sons, New York). These results indicate that inhibition of the chloroplast enzyme takes place at a site other than the 5 active site, which moreover, does not exist in the serine acetyltransferase isoform which is present in the cytosol. These inhibition constants are consistent with the cysteine concentration determined for pea 10 chloroplasts of 40 ± 10 yM (2 nmol/mg chlorophyll), a value which is calculated for a stromal compartment volume of the order of 35 to 65 yl per mg of chlorophyll. 15 Dissociation of the bi-enzymatic complex, cysteine synthase, by cysteine The serine acetyltransferase of the plant cell, like its bacterial homologue, forms an enzymatic complex with 0-acetylserine (thiol) lyase, the enzyme 20 which catalyses the condensation of reduced sulphur with O-acetylserine. This bi-enzymatic complex is called cysteine synthase. All of the serine acetyltransferase of the chloroplast exists in a form complexed with 0-acetylserine (thiol) lyase, while the 25 majority of the O-acetylserine (thiol) lyase is in the free form. The distribution of each of these enzymes in each of the subcellular compartments of pea leaves is described in Table II.

31 Table II: Specific activity of serine acetyltransferase and O-acetylserine (thiol) lyase activities in the cellular compartments of pea leaves Serine aceytl- O-acetylserine transferase (thiol) lyase Specific activity (mU/mg) OASTL/SAT Ratio Stroma 0.85 260 306 Matrix 12 50 4 Cytosol 0.90 180 200 5 The ratio of O-acetylserine (thiol) lyase (OASTL) activity to serine acetyltransferase (SAT) activity reflects the large excess of OASTL over SAT. In particular in the stoma (chloroplast), where the 10 assimilation and reduction of sulphate takes place, and in the cytosol, 95% of the OASTL activity is in the free form. These conditions are necessary for optimal synthesis of cysteine [141. The cysteine synthase complex is composed of a serine acetyltransferase 15 tetramer and two 0-acetylserine (thiol) lyase dimers. O-Acetylserine, the reaction product of serine acetyltransferase, dissociates this bienzymatic complex, and sulphur tends to stabilize it [141. These protein-protein interactions within the complex confer 20 novel properties on each of the enzymes; in particular serine acetyltransferase acquires novel catalytic 32 properties compared to the free form. Moreover, complexed 0-acetylserine (thiol) lyase is inactive in cysteine synthesis, and only the free form (in excess in the cell) catalyses cysteine synthesis [14]. 5 A chloroplast (Pisum sativum) fraction, pre incubated in the presence of an optimal concentration of cysteine (0.1 mM), which inhibits serine acetyltransferase (see Figure 2), then undergoes gel filtration chromatography which allows the separation 10 of molecules according to their molecular mass. Under these conditions the cysteine synthase complex dissociates into serine acetyltransferase tetramers and O-acetylserine (thiol) lyase dimers. Chloroplast serine acetyltransferase in its free form is still sensitive 15 to inhibition by cysteine. To refine this result and to confirm that inhibition of the enzyme is not dependent upon interaction with OASTL, a serine acetyltransferase was partially purified from pea chloroplasts, by ion exchange chromatography followed by molecular 20 filtration chromatography carried out in the presence of O-acetylserine (1 mM), a condition which leads to dissociation of the complex. The serine acetyltransferase fraction thus free of contamination by 0-acetylserine (thiol) lyase 25 is incubated in the presence of increasing concentrations of cysteine under the conditions described in Table I and Figure 2. The calculated IC 50 is of the order of 15 +/- 3 micromolar and is 33 comparable to the value obtained above for the enzyme under chloroplast conditions (see Table I). This latter result makes it possible to establish a model to explain the inhibition of chloroplast serine 5 acetyltransferase. In Figure 3, the tetrameric form of serine acetyltransferase (SAT) is depicted by the grey circles and the O-acetylserine (thiol) lyase (OASTL) dimer by the black circles. The functional cysteine synthase complex in the cell is depicted by the 10 combination of the two molecular populations. In the presence of cysteine, the cysteine synthase complex binds cysteine, which modifies the protein-protein interactions within the cysteine synthase complex, and leads to dissociation into SAT tetramers and OASTL 15 dimers. The SAT thus in its free form is therefore sensitive to cysteine, and it is known that this structure has a tendency to form aggregates (apart from the cysteine synthase complex) whose effect is to cause a loss of its activity [14]. 20 Example 2. Isolation and characterization of a gene encoding a putative cytoplasmic serine acetyltransferase isoform (SAT3) [12] In this example the procedure described on 25 page 502 of Ruffet et al. [12], is taken up, in particular the chapters described under the headings "Bacterial strain and growth conditions" and "Isolation 34 of A. thaliana serine acetyltransferase cDNA clones by complementation in E. coli". A gene encoding a putative cytosolic serine acetyltransferase (Z34888 or L34076) represented in 5 Figure 4 (SEQ ID NO 1), was isolated by functional complementation of an Escherichia coli strain deficient in serine acetyltransferase activity. Analysis of the primary sequence showed the presence of strong similarity with the sequence of the bacterial enzyme 10 (56% homology and 41% identity). The following primers were used to amplify the nucleotide sequence and to clone it into the vector used for transforming tobacco plants: Oligo 1: 5'GAGAGAGGAT CCTCTTTCCA ATCATAAACC ATGGCAACAT GCATAGACAC ATGC 3' Oligo2: 5'GGCTCACCAG ACTAATACAC TAAATTGTGT TTACCTCGAG AGAGAG 3' 15 These primers make it possible to introduce a 5' BamH1 restriction site (GGATCC) and a 3' Sacl restriction site (GAGCTC). The N terminus of the amino acid sequence of the SAT3 isoform does not have the characteristics of 20 organelle (mitochondrion or chloroplast) addressing peptides. This analysis leads to the assumption that this isoform is located in the cytosol [12]. The absence of an addressing peptide of chloroplast type in th-is isoform was confirmed in chloroplast import 25 experiments ([29] Murillo et al. 1995, Cell. and Mol.

35 Biol. Research 41, 425-433). Conversely, a study using constructs which include a portion of the nucleotide sequence and a marker protein (Green Fluorescent Protein, GFP) showed the presence of the fusion product 5 (5'-SAT3-GFP) in the chloroplast of transformed A. thaliana plants (vegetative stage of the plant) and also in the cytosol (at the floral stage)[27]. The SAT3 gene (L34076) contains no introns. 10 Example 3. Overexpression and purification of SAT3 in Escherichia coli The defined protocol for overexpression of the enzyme in E. coli makes it possible to purify the enzyme in its free form or complexed with plant 15 0-acetylserine (thiol) lyase, the cysteine synthase complex [14]. Using the purified proteins, the effect of cysteine on serine acetyltransferase activity was analysed by a spectrophotometric assay based on the consumption of acetyl-CoA during reaction 1, as a 20 function of incubation time. This analysis is carried out in a medium (1 ml) containing 50 mM Hepes-HCl, pH 7.5, 2 mM L-serine and 0.2 mM acetyl-CoA. The reaction is followed by measuring the decrease in absorbance at 232 nm (molecular extinction coefficient of 25 4200 M- 1 cm- 1 )([30] Kredich, N.M. et al., J. Biol. Chem. (1969) 244, 2428-2439). We were able to show that this isoform (SAT3) in its free form or complexed with 0-acetylserine (thiol) lyase, is cysteine-insensitive.

36 This result allows us to confirm that this cDNA (L34076, Figure 4) encodes a cytosolic serine acetyltransferase, since the amino acid composition of the N-terminus does not have the characteristics of 5 transit peptides, and moreover, since this serine acetyltransferase is cysteine-insensitive. This latter result is similar to observations which have been obtained for the cytosolic serine acetyltransferase activity of pea leaves (Figure 2 and Table I). 10 Example 4. Isolation and characterization of a gene encoding a cytoplasmic serine acetyltransferase isoform (SAT3')(U30298) The procedure of Example 3 is repeated, using 15 oligonucleotides 3 and 4 below: Oligo 3: 5' GAGAGAGGAT CCTCTTATCG CCGCGTTAAT ATGCCACCGG CCGGAGAACTC C 3' Oligo4: 5'GAGCCTTACC AGTCTAATGT AGTATATTTC AACCTCGAGA GAGAG 3' A gene is isolated which encodes an acetyltransferase (U 30298), and is represented in Figure 5 (SEQ ID NO 2). Analysis of the primary 20 sequence showed the presence of strong similarity with the sequence of the bacterial enzyme (51.6% homology and 42.6% identity). The N-terminal structure (absence of the conditions necessary for organelle addressing) indicates that this isoform is located in the cytosol. 25 On the other hand, it is given as being cysteine- 37 sensitive [27]. This result differs from the data obtained from pea leaves (and from spinach leaves), in the sense that the cysteine regulation site seems to be confined to the cytosol in A. thaliana [27]. Moreover, 5 it would seem that A. thaliana has at least two cytosolic isoforms: SAT3 (Example 3) and SAT3' (or U30298, Example 4). Unlike the SAT3 gene, the gene corresponding to SAT3' has an intron. 10 Example 5. Isolation and characterization of genes encoding a serine acetyltransferase isoform (SAT1') The procedure described in Example 3 is repeated for the present example. A gene encoding a serine acetyltransferase 15 (L78443), which is represented in Figure 6 (SEQ ID NO 3), was isolated by functional complementation of an Escherichia coli strain deficient in serine acetyltransferase activity [12]. Analysis of the primary sequence shows strong similarity with the 20 sequence of the bacterial enzyme (52.7% homology and 39% identity). The following primers were used to amplify the nucleotide sequence and to clone it into the vector which is used for transforming tobacco plants (in bold 25 characters in Figure 3): 38 Oligo 5: 5'GAGAGAGGAT ._CCTCCTCC TCCTCCTCCT ATGGCTGCGT GCATCGACAC CTG 3' Oligo6: 5'GCTCACCAGC CTAATACATT AAACTTTTTC AGCTCGAGAG AGAG 3' These primers make it possible to introduce a 5' BamH1 restriction site (GGATCC) and a 3' Sac1 restriction site (GAGCTC). 5 A second gene is obtained which encodes a putative mitochondrial serine acetyltransferase (U22964), and is represented in Figure 7 (SEQ ID NO 4), by repeating the same procedure, using oligo 7 to replace oligo 5 as the 5' primer. Oligo 7*: 5'GAGAGAGGAT CCGGCCGAGA AAAAAAAAAA ATGTTGCCGG 10 TCACAAGTCG CCG 3' The N-terminus of the amino acid sequence of the SATl isoform has the characteristics of organelle (mitochondrion or chloroplast) addressing peptides. Localization in the mitochondrion was recently 15 confirmed by constructing a fusion protein which includes the 5' portion and "green fluorescent protein" (5'SAT1-GFP) and by transforming Arabidopsis thaliana plants [27]. Neither the SATl' gene (L78443) nor the SAT1 gene (U22964), like its homologue (SAT3), has 20 introns.

39 Example 6. Overexpression and purification of SAT1 in Escherichia coli. Localization of this isoform in A. thaliana The defined protocol for overexpression of 5 the enzyme in E. coli makes it possible to purify the enzyme (in its transit peptide-lacking form, SAT L78443) in its free form or complexed with plant O-acetylserine (thiol) lyase, the cysteine synthase complex (14]. Using the purified proteins, the effect 10 of cysteine on serine acetyltransferase activity was analysed by spectrophotometric assay, based on the consumption of acetyl-CoA during reaction 1, as a function of incubation time (see Example 3). Analysis was also carried out by HPLC assay of the reaction 15 product (OAS) (see Example 1). We were able to show that this isoform (SAT1'), in its free form or complexed with O-acetylserine (thiol) lyase, is cysteine-insensitive. This latter result parallels the observations obtained for pea leaf mitochondrial serine 20 acetyltransferase activity (Figure 2 and Table I), the latter being inhibited at non-physiological concentrations of cysteine. Using a preparation of mitochondria obtained from pea leaves or from protoplasts from cell cultures, 25 localization in the mitochondrion was confirmed for this isoform. A mitochondrial fraction lacking in plastid and in cytosolic contaminants was obtained by using the 40 protocol defined for pea leaf mitochondria [12). The molecular mass of the polypeptide as revealed by antibodies raised against the peptide [-TKTLHTRPLLEDLDR-] (see SATl amino acid sequence), is 5 of the order of 34,000 daltons, a value which is in agreement with the mass of the protein as obtained using sequence analysis programs for predicting cleavage sites. 10 Example 7. Isolation and characterization of genes encoding a serine acetyltransferase isoform (SAT2) The procedure described for Example 3 is repeated for the present example. A gene which encodes a serine 15 acetyltransferase (L78444), represented in Figure 8 (SEQ ID NO 5), was isolated by functional complementation of an Escherichia coli strain deficient in serine acetyltransferase activity [12]. Analysis of the primary sequence showed the presence of strong 20 similarity with the sequence of the bacterial enzyme (49.5% homology and 35.4% identity). The following primers were used to amplify the nucleotide sequence and to clone it into the vector which was used to transform tobacco plants (in bold 25 characters in Figure 8): 41 Oligo 8 5' GAGAGAGGAT CCGACAAGTT GGCATAATTT ATGGTGGATC TATCTTCCT 3' Oligo 9 5' CCTGTGTGAT TGTCGTGTAG TACTCTAGAA ACTCGAGAGA GAG 3' These primers make it possible to introduce a 5' BamH1 restriction site (GGATCC) and a 3' Sac1 restriction site (GAGCTC). 5 Analysis of the N-terminal portion of the sequence shows the presence of characteristics for addressing of the protein to an organelle (mitochondrion or chloroplast). Unlike the other isoforms described above, the SAT2 gene is complex and 10 has several introns. Comparing SAT2 sequences with its homologues from A. thaliana, from plants and from other organisms, leads to the assumption of a prokaryotic origin (Figure 10). Moreover, analysis of the N-terminal sequence using the chloroP program 15 [http://www.cbs.dtu.dk/services/chlorP/], indicates a high probability of the presence of a chloroplast-type transit peptide. Example 8. Isolation and characterization of genes 20 encoding a serine acetyltransferase (SAT4) isoform A gene which encodes a serine acetyltransferase (SAT4), represented in Figure 9 (SEQ ID NO 6), was isolated by functional complementation of an Escherichia coli strain deficient in serine 25 acetyltransferase activity [12]. Analysis of the 42 primary sequence showed the presence of strong similarity with the sequence of the bacterial enzyme (44.5% homology and 32% identity). The following primers were used to amplify 5 the nucleotide sequence and to clone it into the vector which was used for transforming tobacco plants: Oligo 10 :5'GAGAGAGGAT CCGACAAGTTGG CATAATTTAT GGCTTGTATA AACGGCGAGA ATCGTGATTT TTCTT 3' Oligo11: 5'TACCTCGTAC CACTCAGAAC TCTAGAAACT CGAGAGAGAG3' These primers make it possible to introduce a 5' BamH1 restriction site (GGATCC) and a 3' Sac1 10 restriction site (GAGCTC). Analysis of the N-terminal portion sequence shows the presence of characteristics for addressing of the protein to an organelle (mitochondrion or chloroplast). The SAT4 gene, like that of SAT2, is 15 complex and has several introns. Comparing SAT4 sequences with its homologues from A. thaliana, from plants and from other organisms, leads to the assumption of a prokaryotic origin (Figure 10). Moreover, analysis of the N-terminal sequence using the 20 chloroP program [http://www.cbs.dtu.dk/services/chlorP/], indicates a high probability of the presence of a chloroplast-type transit peptide. Figure 10 represents the sequence comparison and was carried out using the Clustaw 25 program (Vector NTI software). SAT2 and SAT4 are closer to the prokaryotic SATs than are SAT3, SATI and SAT52.

43 Moreover, the branch also comprises an SAT from red alga (AB00848), which has been identified as a cysteine-sensitive protein located in the chloroplast ([32] Toda et al., 1998, Biochim. Biophys. Acta 1403, 5 72-84). SAT4 is identified as being on chromosome 4 (Bac clone F8D20, access number AL031135). Example 8. Constructs used for transforming tobacco plants of the small Havanna variety 10 Transgene expression in leaves Transformation of tobacco plants is carried out through Agrobacterium tumefaciens EHA105, which contains the pBIl21 vector (Clontech) (Figures 11 and 12). 15 SAT3 (or SAT1' or any cysteine-insensitive SAT) To obtain expression of the SAT3 (SEQ ID NO 1) of Example 2 in the chloroplast (Figure 11), an extension which allows addressing to this compartment 20 is introduced 5' of the cDNA. For this, the optimized transit peptide previously described is used. A kanamycin-resistance gene (NPTII) which encodes neomycin phosphotransferase, and which is used as a selection marker for transforming tobacco, is 25 cloned between the left (LE) and right (RE) edges of the t-DNA. Expression of the NPTII gene is under the control of the promoter and of the terminator of A. tumefaciens nopalin synthase. Downstream, the 44 $-glucuronidase gene which has been cloned between the unique BamHl and the unique Sac1 sites, is under the control of the cauliflower mosaic virus (CaMV) 35S promoter and the nopalin synthase gene polyadenylation 5 signal from the Ti plasmid. The OTP-SAT3 construct is inserted between the Xho and Sacl sites of the vector, from which has been deleted the $-glucuronidase gene (Figure 11). SAT1, SAT3, SAT3', SAT2, SAT4 or any SAT 10 To obtain SAT expression in any of the subcellular compartments (cytosol, mitochondrion or chloroplast), the transgene is introduced into the appropriate vector, which is described in Figure 12. A kanamycin-resistance gene (NPTII) which 15 encodes neomycin phosphotransferase, and which is used as a selection marker for transforming tobacco, is cloned between the left (LE) and right (RE) edges of the t-DNA. Expression of the NPTII gene is under the control of the promoter and of the terminator of 20 A. tumefaciens nopalin synthase. Downstream, the $-glucuronidase gene which has been cloned between the unique BamH1 and the unique Sac1 sites, is under the control of the cauliflower mosaic virus (CaMV) 35S promoter and nopalin synthase gene polyadenylation 25 signal from the Ti plasmid. The gene encoding the SAT is inserted between the BamHl1 and Sac1 sites of the vector, from which has been deleted the S-glucuronidase gene (Figure 12).

45 Transgene expression in seeds A construct similar to that shown in Figures 11 or 12 is prepared with the aim of obtaining specific expression of the transgene in the seeds. This strategy 5 may be important since seeds make up the main contribution to the animal diet. For this, the constitutive tobacco mosaic promoter is replaced with a promoter which allows specific expression of the transgene during the setting up of the seeds' stocks. 10 Example 9. Transformation of tobacco Young leaves of tobacco plants (aged from 3 to 4 weeks) whose surface is sterilized with a 10% (V/V) solution of bleach for 10 min then rinsed with 15 sterile water, are cut up with a punch (30 discs per construct). 20 ml of a 48-hour culture of Agrobacterium tumefaciens EHA105 (containing the pBI121 vector modified according to the invention) are centrifuged and then resuspended in 4 ml of a 10 mM solution of 20 MgSO 4 . The foliar discs are incubated for a few minutes in the solution of agrobacteria, then transferred to MS medium (Sigma M-5519) supplemented with 0.05 mg/l of a-naphthaleneacetic acid (NAA, Sigma), 2 mg/l of 6-benzylaminopurine (BAP) and 7 mg/l of phytoagar, for 25 2 to 3 days. The foliar discs are then transferred to an identical medium to which are added 350 mg/l of cefotaxin (bacteriostatic) and 75 mg/l of kanamycin (selection agent). After 2 weeks, discs on which have 46 developed calli as well as young shoots, are subcultured in identical medium in order to accelerate growth of the shoots. A week later, the green shoots are excised and transferred into the same medium, 5 without hormone, in order to allow the development of roots, this for about 2 weeks, at the end of which the young plants are transferred into earth and cultivated in a hothouse. 10 Example 10. Analysis of results for SAT3 and SATl' (L78443) (truncated form of the SATl U22964) transgenic plants and controls The impact of the expression of SAT3, SAT1' or OTP-SAT3 in leaves or in seeds of tobacco plants is 15 analysed as regards the content of sulphur compounds; cysteine, methionine (and derivatives such as S-methylmethionine or SMM) and glutathione. The cysteine and glutathione are evidenced by the method of Fahey ([33] Fahey, R.C. and Newton, G.L. Methods 20 Enzymol. (1987) 143, 85-96), after derivatization of the compounds by thiolyte (mBBR from Calbiochem) and separation by high performance liquid chromatography (HPLC) [33]. The free methionine and SMM are assayed by the methods for assaying free amino acids after 25 extraction, derivatization with ortho-phthalaldehyde, and separation by HPLC ([34] Brunet, P. et al., J. Chrom. (1988) 455, 173-182). The serine acetyltransferase activity is measured as described in 47 the methodology for assay of formed O-acetylserine, by the HPLC technique, or by the method of coupling in the presence of an excess of O-acetylserine (thiol) lyase [12], [14]. The SAT transgene activity in transformed 5 plants (i.e. in vivo) is revealed by assaying the O-acetylserine, which is produced during activity of the enzyme and is transiently accumulated in the cell. The O-acetylserine in the plant extracts is assayed following the protocol below. 10 After crushing tobacco leaves to a fine powder in liquid nitrogen, the extracts are taken up in 0.1 M hydrochloric acid (1 ml/100 mg of powder). After an incubation period of about 10 min, the debris is eliminated by centrifugation for 15 min at 15,000 g. A 15 fraction of the obtained supernatant, containing the free amino acids, is derivatized for 1 min at 25 0 C in the presence of a solution of ortho-phthalaldehyde (solution containing 54 mg of ortho-phthalaldehyde, 10% methanol, 90% sodium borate, 400 mM, pH 9.5, and 0.2 ml 20 of $-mercaptoethanol). The OPA-amino acid derivatives are separated by reverse phase chromatography on a UPHDO-15M column (0.46 x 150 mm - Interchim) connected to an HPLC system (Waters). The buffers used to carry out the elution are, buffer A: 85 mM sodium acetate, pH 25 4.5 supplemented with acetonitrile to 6% final; buffer B: 60% acetonitrile in water. Separation of the derivatives is carried out according to the gradient (1 ml/min): 0 min, 30% B in A; 8 min, 60% B in A; 48 9 min, 80% B in A; 10 min, 100% B; 12 min, 100% B. At the column exit, the fluorescence emitted by the derivatives is measured at 455 nm after excitation at 340 nm (SFM25 fluorimeter, Kontron). 5 The retention time of O-acetylserine under our experimental conditions is 9.5 min. The identity of the peak corresponding to 0-acetylserine is confirmed by co-elution with a known quantity of the pure product. Moreover, a second control is carried out to 10 confirm the position of O-acetylserine in the various analyses. The samples, before incubation with OPA, are treated with NaOH at a final concentration of 0.2 M. Under these conditions, the acetate group in the OH position on serine is transferred to the amine group, 15 thus allowing the formation of N-acetylserine. This latter compound is no longer detected under our experimental conditions and thus leads to the disappearance of the peak which initially corresponded to 0-acetylserine. 20 Plants transformed with an SAT transgene were preselected with kanamycin, and run to seed. Control plants (PBI, three independent lines which contain the transforming vector and a GUS cassette) are treated in an identical way. Analyses of the plants comprise: 1; 25 demonstration of insertion of the transgene into the genome by PCR, using the 5' primer and the 3' primer which correspond to the SAT which is used for the transformation; 2, demonstration of the messenger by 49 analysis of messengers using probes which correspond to the SAT transgenes used for transforming the plants according to known techniques; 3, demonstration of enzyme activity associated with SAT protein according 5 to methods described in the literature [14], and demonstration of transgene localization; 4, assay of the product of the SAT reaction, i.e. O-acetylserine (OAS), in transformed plants; 5, assay of cysteine and its direct derivatives, of glutathione and of 10 methionine (and its methylated derivatives); 5, analysis of total amino acid composition of the plants and seeds which are associated with each of the transgenes obtained (free amino acids and amino acids linked to proteins), according to traditional 15 techniques; 6, analysis of the impact of overexpressing SAT activity in plant cells, on the amount of enzyme activity which is associated with the sequence of assimilation of sulphur (sulphate transporters, ATP sulphurylase, APS reductase, sulphite reductase and in 20 particular O-acetylserine (thiol) lyase, the enzyme which is directly associated with SAT activity in cysteine synthesis (14]. Moreover, the enzymes associated with the synthetic pathway of methionine and the synthetic pathway of glutathione, are analysed in 25 order to understand the impact of the cysteine content on the metabolism associated with glutathione synthesis and methionine synthesis.

50 Expression of the Arabidopsis thaliana serine acetyltransferase gene in tobacco leads to an increase in the level of cysteine, the level of glutathione and the level of methionine in tissues of transformed 5 plants, compared to control plants. In general, this increase in the amount of free sulphur compounds is associated with transgene expression in the plant cell (Figure 13). Measurement is carried out on leaves from 3 different plants for each homozygous line. The SAT 10 activity is measured as its capacity to promote cysteine synthesis, according to the protocol described above [14]. Expression of the transgene under the control of the constitutive CaMV promoter, causes the SAT 15 capacity (maximum potential enzyme activity measured in vitro) to increase by a factor of 2 to 8, compared to the level measured in control plants (plants transformed with an empty vector). To determine the real activity of the SAT transgene, the amount of 20 0-acetylserine (free OAS) was measured. Thus, it was possible to multiply the level of OAS in plant cells (average level of 4 nmol/g of fresh material for control plants, 6 independent measurements) by a factor of 2 to 10, in transformed plants (2 independent 25 measurements). Thus, for most SAT transgenes, associated with the clear increase in the capacity of SAT enzyme activity, is an increase in free intracellular OAS which results from the transgene 51 activity in vivo, and an increase in the amount of free cysteine, compared to control plants (Figure 14). The cysteine content in the control plants (PBI) and in the T2 tobacco plants transformed with an SAT (SATl' and 5 SAT3 lines), is determined as monobromobimane derivatives, by HPLC, for 3 plants per line [33]. The cysteine content of the transgenic lines is increased 2- to 10-fold in comparison with control plants (PBI). The amount of free cysteine in most 10 transgenic plants which express an SAT is significantly higher, 2 to 10-fold, than the natural level which is measured in control plants PBI (of a value of 5 nmol/g of fresh material, average calculated from three independent lines, each containing 5 plants). This 15 impact of SAT expression is observed as early as the T1 generation. On the other hand, no correlation could be seen between amount of cysteine (and moreover of free OAS) and the SAT activity from transgenes which are measured in vitro. On the other hand, a significant 20 positive correlation could be measured between amount of cellular OAS and cysteine level in the cell (Figure 15). In vivo, a 3- to 10-fold increase, compared to control plants, in the level of free O-acetylserine, which is linked to transgene activity, 25 results in a 3- to 8-fold increase in the level of cysteine in the plants. Analysis was carried out on fully developed leaves (about 2 months) of plants homozygous for the transgene. The control plants are 52 plants transformed with empty constructs (PBI). An increase in the amount of free cellular OAS which is linked to SAT transgene activity in transformed plants, correlates positively with increase in the amount of 5 cysteine. Thus, an average 6-fold increase in the level of free OAS is associated with a 6-fold increase in the level of cysteine. The slope associated with the distribution of the points is 1.06 +/- 0.09 (coefficient of regression 0.67). It indicates that for 10 each molecule of OAS accumulated, one mole of cysteine is synthesized. The value of this slope and the absence of a plateau observed under our experimental conditions, indicate the sulphide formation (assimilation of sulphate and reduction to sulphide) is 15 not a limiting pathway and that SAT activity seems to be the limiting factor in the cell for cysteine formation (Figure 1). The subcellular localization of the SAT1' (truncated form of SAT1) transgene and the SAT3 20 transgene in transformed tobacco plants was made clear by preparation of the chloroplast fraction of transformed plants which present the highest enzyme activity, compared with PBI plants (controls). The activity associated with the chloroplast compartment is 25 compared with that measured in the total extract (Figure 16). The values for serine acetyltransferase activity correspond to 3 lines for the PBI plants (5 plants per line), to 5 lines for SAT1' and SAT3, each 53 being represented by 5 plants. The columns in grey correspond to the activities measured in the total extract from each of the lines, and the columns in black represent the average of the activities measured 5 in each of the chloroplast preparations. These results establish definitively that SAT3 is an isoform of the serine acetyltransferase located in the cytosol of plant cells, and that the truncated form of SATl (absence of transit peptide) is 10 also located in the cytosolic compartment. With regard to SAT3, these results confirm our interpretations which are derived from analysis of the protein sequence [12]. A direct consequence of increasing the level 15 of cellular cysteine is increased synthesis of glutathione and methionine (see Figure 1). Cysteine is destined for multiple usage and besides its incorporation into proteins, and its participation in the synthesis of multiple compounds, such as vitamins 20 (biotin, thiamine, etc. and other sulphur derivatives in the cell), cysteine also participates in the synthesis of glutathione (tripeptide which is associated with numerous plant defence mechanisms and which is considered to be a reservoir for cysteine) and 25 of methionine. Specifically in plants which are transformed with the SAT transgene, the level of glutathione correlates directly with that of cysteine, and is reflected by an increase of 2 to 7 times the 54 natural level which is measured in control plants (PBI) (Figure 17). The correlation coefficient which is calculated for the distribution of the points is 0.92. A 4-fold increase in cysteine content in transgenic 5 tobacco plants which overexpress SAT results in a 3- to 4-fold increase in the level of glutathione. Analysis was carried out using fully developed leaves (about 2 months) from plants homozygous for the transgene. The control plants are plants which are transformed with 10 empty constructs. This result indicates that cysteine is the limiting factor in glutathione synthesis in the plant cell. Thus, indirectly, the consequence of any modification of the level of serine acetyltransferase 15 in the cell, will be to increase the amount of intracellular glutathione, by increasing the level of cysteine. This result implies that the transgenic plants obtained have acquired properties of stress resistance compared to the control plants (PBI). This 20 aspect was observed recently ([34] Blaszczyl A. et al., 1999, The Plant Journal 20, 237-243). Moreover, the amount of cysteine and of glutathione which is considered to be a reservoir, brings about increased availability at the time of synthesis of polypeptides 25 rich in cysteine (for example for resistance to phytopathogenic attack), and rich in cysteine and in methionine (for animal foods).

55 An increase in cysteine in the plant cell also brings about an increase in the relative amount of methionine (Figure 18). On the other hand, unlike the results observed for glutathione, the curve has a 5 plateau, which seems to indicate the existence of another control site which would limit methionine synthesis. Moreover, homocysteine, which is derived from the trans-sulphuration pathway, and is the sulphur precursor in cysteine synthesis, does not seem to 10 accumulate. This observation thus indicates that the folate pool in the plant cell, which is essential for methylation and for methionine formation, is not a limiting factor. This limitation would thus be situated downstream of cysteine and upstream of homocysteine. It 15 concerns the synthesis of the carbon precursor for the aspartate-derived methionine synthesis (0-phosphohomoserine and/or cystathionine). The level of aspartokinase (the first enzyme of the aspartate pathway for the synthesis of lysine, threonine and 20 methionine) is controlled by several effectors, such as threonine and S-adenosylmethionine (SAM) which comes from methionine synthesis [3]. Cystathionine y-synthase (see Figure 1) is directly regulated at the transcriptional level [3] and, more exactly, methionine 25 or one of its derivatives controls the stability of its messenger [4]. The maximum plateau which is obtained under our experimental conditions is of the order of 39 +/- 7 nmol of methionine/g of fresh material, which 56 corresponds to a multiplication of the average natural level which is of the order of 6 +/- 2 nmol per g of fresh material (PBI control). The maximum value which is obtained for methionine requires an increase in the 5 amount of cysteine in the cell of 4 to 5 times its maximum level. The regression coefficient is 0.50. Moreover, an increase in the methionine in the cells causes the level of S-methylmethionine (SMM) to multiply from 2- to 10-fold, according to the plant. 10 SMM is derived directly from the methylation of methionine in the presence of S-adenosylmethionine. This compound is important to the cell, and is a form of transport of methyl groups (of methionine) in the plant. In the presence of one molecule of homocysteine 15 (the sulphur precursor in methionine synthesis, and which is derived from cysteine), SMM allows the synthesis of two molecules of methionine ([3], [35], Bourgis et al., 1999, Plant Cell 11, 1485-1497). It may thus have a primordial role at the time of storage 20 protein synthesis in the seed. Moreover, SMM is the direct precursor for the synthesis of compounds such as 3-dimethylsulphoniopropionate which is involved in the resistance of plants to salt stress ([36] Hanson A.D. et al., 1994, Plant Physiol. 105, 103-110). Such an 25 approach has many consequences, in particular for increasing the potentialities of plants on grounds rich in salt.

57 Evidence for a regulatory role in the sulphate assimilation pathway in vivo. Serine acetyltransferase is taken to be a limiting factor for the assimilation of sulphur and for 5 the synthesis of cysteine. Its role in bacteria is important since the reaction product, (0-acetylserine, OAS) or its derivative (N-acetylserine), is the effector which modulates the expression of the genes of the sequence of assimilation of sulphur, such as: 10 1, sulphate transport, 2, ATP sulphurylase, 3, APS kinase, and 4, PAPS reductase ([37] Kredich N.M., 1987, in Escherichia coli and Salmonella typhimurium: cellular and molecular biology, pp. 419-428). In plants, a role has been shown for OAS in modulating the 15 expression of several genes, which concerns sulphate transporters, ([38] Smith F.W. et al., 1997, The Plant Journal 12, 875-884; [39] Hawkesford M.J. et al. 1995, Z. Pfanzenerndrh. Bodenk. 158, 55-57; [40] Clarkson D.T. et al. 1999, Plant Physiol. Biochem. 37, 283-290), 20 ATP sulphurylase [39-40] and APS reductase ([41] Neuenschwander U. et al. 1991, Plant Physiol., 97, 253 258). The role of serine acetyltransferase activity in gene modulation has been proposed based on the kinetics of the cysteine synthase complex (bienzyme complex 25 composed of serine acetyltransferase and of O-acetylserine (thiol) lyase) ([41] Droux et al. in Sulphur and Nutrition in Plants, in press), and has led to the description of a model to describe the mechanism 58 of gene regulation. The role of OAS is also determinant in the regulation of gene expression during seed formation ([42] Kim H. et al., 1999, Planta 209, 282 289). 5 In transgenic plants which overexpress an SAT in the cytosol, a transient increase in OAS was shown (increase of 2 to 10 times its natural level, see Figure 15). In parallel, in most transgenic plants, an increase in OASTL activity was measured (Figure 19). 10 This increase of 2 to 5 times compared to the activity which is measured in PBI controls, concerns only the chloroplast-associated activity. Moreover, in a Western Blot, the signal which is observed is stronger in most transgenic lines (Figure 20), indicating that the 15 increase in activity corresponds to an induction of de novo synthesis of OASTL. This original result corresponds to the first demonstration of the role of OAS (in planta) in the modulation of genes of the sulphate assimilation pathway, in particular for 20 chloroplast OASTL. Referring to Figure 20, an equivalent amount of protein (0.150 mg) undergoes SDS-PAGE (12%), and after separation, the proteins are transferred onto a nitrocellulose membrane. The presence of OASTL is 25 revealed by incubation with antibodies which have been raised against chloroplast OASTL from spinach leaves [7].

59 Overexpression of SAT in plant cells thus causes the capacity to synthesize cysteine in the chloroplast to increase. It can, therefore, be assumed that the expression of genes encoding enzymes of the 5 sulphate assimilation and reduction pathway (sulphate transporter, ATP sulphurylase, APS reductase, sulphite reductase) is also modulated like OASTL (and references [38-411). The increase in the intracellular content of 10 OAS (which is derived from SAT activity) signals a state of artificial sulphur stress (absence of sufficient reduced sulphur) in the cell (in transformed plants), which leads to induction of the enzymes of the sulphate assimilation pathway. 15 Impact of increasing cysteine in a cell on the general content of amino acids. This increase in sulphur compounds is accompanied by an increase in the content of essential amino acids, such as threonine, isoleucine 20 and lysine (their amount is doubled, on average). On the other hand, the level of glutamate is halved, as is that of aspartate. This latter observation is directly linked to the increase in the amount of THR, LYS and ILE. All the increases in amino acids correlate with an 25 increase in serine acetyltransferase (SAT3 or SATl') activity in the cytosol. Moreover, an increase in these sulphur compounds leads to an improvement in the nutritional ratio N/S of the plant (on the basis of 60 free amino acids). It is reflected by a drop in this relative ratio, due to the enrichment in total sulphur compounds (cysteine, methionine, SMM and glutathione). This factor is important since it conditions the 5 polypeptide content of the seeds, and leads to enrichment (or impoverishment if the N/S ratio is too high) of storage proteins whch are rich in sulphur containing amino acids, to the detriment of polypeptides which are lacking in these compounds. 10 Example 11. Analysis of OTP-SAT3 (OTP-SAT1') transgenic plants Analysis of transformants at the TO stage of transgenic plants which express a cysteine-insensitive 15 SAT (here for example, SAT3 or SATl'; truncated form of SAT1 U22964), in leaves or in seeds (under the control of a seed-specific promoter), reveals an increase in free cysteine content, but also in glutathione content (2.6 times the natural level), and in methionine 20 content. Plants which express these same isoforms in the cytosol under the control of a seed-specific promoter show a level of sulphur compounds which is higher that in control plants.

61 Example 12. Analysis of results for SAT1 (cDNA U22964 or SAT1jw, transit peptide form) transgenic plants and control plants. The impact of expression of serine 5 acetyltransferase in mitochondria was analysed by transforming plants with the construct (Figure 12) which contains the entire SAT1 sequence. Analysis of plants at the TO stage makes it possible to show an increase in free cysteine in the cell (Figure 21). 10 Analysis is carried out on leaves which are formed before appearance of the floral scape. The fourteen lines show a 2- to 6-fold multiplication in cysteine level, compared with the control plant (PBI). The increase in cysteine is accompanied by a 15 general effect on the amount of sulphur compounds, with a 4-fold multiplication in the amount of glutathionine in the cell (Figure 22). Unlike the case of SAT expression in the cytosolic compartment, the general appearance of the distribution of 'values in the 20 different lines, shows a plateau which would indicate limitation in glutathione synthesis. This limitation may concern the level of glutamate and/or glycine or may concern glutathione control of its own synthesis (retroinhibition of one of the enzymes which 25 participate in glutathione synthesis, enzyme E6 and/or enzyme E7 see Figure 1).

62 Similarly, the amount of methionine is multiplied 2- to 3-fold compared to the natural level which is measured in control plants.

Claims (47)

1. Method for increasing the production of cysteine, glutathione and methionine, and of sulphur derivatives thereof, by plant cells and plants, the 5 said method consisting in overexpressing an SAT in plant cells and plants containing the said plant cells.

2. Method according to claim 1, characterized in that the SAT which is overexpressed in plant cells is a cysteine-sensitive SAT. 10 Method according to claim 2, characterized in that the SAT is a plant SAT or a native SAT of bacterial origin.

4. Method according to claim 1, characterized in that the SAT which is overexpressed in 15 plant cells is a cysteine-insensitive SAT.

5. Method according to claim 4, characterized in that the SAT is a plant SAT or an SAT of bacterial origin, or a mutated plant SAT, rendered cysteine-insensitive by mutagenesis. 20 6. Method according to one of claims 1 to 5, characterized in that the SAT is overexpressed in the cytoplasm of plant cells.

7. Method according to claim 6, characterized in that the SAT is an SAT of bacterial 25 origin. 64

8. Method according to claim 6, characterized in that the SAT is a plant cytoplasmic SAT, in particular from Arabidopsis thaliana.

9. Method according to claim 8, 5 characterized in that the SAT is SAT3 which is represented by SEQ ID NO 1.

10. Method according to claim 6, characterized in that the SAT is a non-cytoplasmic plant SAT from which has been removed its signal(s) for 10 addressing to cellular compartments other than the cytoplasm.

11. Method according to claim 10, characterized in that the SAT is SATl' which is represented by SEQ ID NO 2. 15 12. Method according to one of claims 1 to 5, characterized in that the SAT is overexpressed in mitochondria.

13. Method according to claim 12, characterized in that the SAT is overexpressed in the 20 cytoplasm in the form of a signal peptide/SAT fusion protein, the mature functional SAT being released inside mitochondria.

14. Method according to claim 13, characterized in that the mitochondrial addressing 25 signal peptide consists of at least one signal peptide from a natural plant protein which is located in mitochondria, such as for example, the SATl signal 65 peptide which is represented by amino acids 1 to 63 in SEQ ID NO 3.

15. Method according to claim 13, characterized in that the SAT is a mitochondrial SAT of 5 plant origin, in particular from Arabidopsis thaliana.

16. Method according to claim 15, characterized in that the SAT is SATl which is represented by SEQ ID NO 3.

17. Method according to claim 6, 10 characterized in that the SAT is overexpressed in chloroplasts of plant cells.

18. Method according to claim 17, characterized in that the SAT is overexpressed in chloroplasts by integration, into chloroplast DNA of 15 plant cells, of a chimeric gene comprising a DNA sequence encoding the said SAT, under the control of 5' and of 3' regulatory elements which are functional in chloroplasts.

19. Method according to claim 17, 20 characterized in that the SAT is overexpressed in the cytoplasm in the form of a transit peptide/SAT fusion protein, the mature functional SAT being released inside chloroplasts.

20. Method according to claim 19, 25 characterized in that the SAT is homologous with the transit peptide. 66

21. Method according to claim 20, characterized in that the SAT is a chloroplast SAT of plant origin, in particular from Arabidopsis thaliana.

22. Method according to claim 21, 5 characterized in that the SAT is SAT2 or SAT4 which are represented by SEQ ID NO 5 or NO 6, respectively.

23. Method according to claim 19, characterized in that the SAT is heterologous with the transit peptide. 10 24. Method according to claim 13, characterized in that the SAT is a cytoplasmic SAT of plant origin or an SAT of bacterial origin, as defined in one of claims 3 to 5 or 9 to 11.

25. Method according to either of claims 23 15 and 24, characterized in that the transit peptide is a transit peptide from another protein which is located in plastids.

26. Method according to claim 25, characterized in that the transit peptide consists of a 20 plant EPSPS transit peptide or a plant RuBisCO ssu transit peptide.

27. Method according to either of claims 25 and 26, characterized in that the transit peptide comprises a transit peptide from a plant protein which 25 is located in plastids, and, between the C-terminal portion of the transit peptide and the N-terminal portion of the SAT, a portion of sequence from the 67 mature N-terminal region of a protein which is located in plastids.

28. Method according to claim 27, characterized in that the portion of sequence comprises 5 generally less than 40 amino acids from the N-terminal portion of the mature protein, preferably less than 30 amino acids, more preferably between 15 and 25 amino acids.

29. Method according to either of claims 27 10 and 28, characterized in that the transit peptide comprises, between the C-terminal portion of the N-terminal portion of the mature protein and the N-terminal portion of the SAT, a second transit peptide from a plant protein which is located in plastids. 15 30. Method according to claim 29, characterized in that the transit peptide is an optimized transit peptide (OTP) made by fusing a first transit peptide with a portion of sequence from the mature N-terminal region of a protein which is located 20 in plastids, which is fused with a second transit peptide.

31. Transit peptide/SAT fusion protein, characterized in that the SAT is heterologous with the transit peptide. 25 32. Fusion protein according to claim 31, as defined in claims 24 to 30. 68

33. Nucleic acid sequence encoding a transit peptide/SAT fusion protein according to either of claims 31 and 32.

34. Chimeric gene comprising a coding 5 sequence as well as heterologous 5' and 3' regulatory sequences, which are able to function in a host organism, characterized in that the coding sequence comprises at least one nucleic acid sequence which encodes an SAT. 10 35. Chimeric gene according to claim 34, characterized in that the host organism is chosen from bacteria, for example E. coli, yeasts, in particular of the genera Saccharomyces, Kluyveromyces or Pichia, fungi, in particular Aspergillus, baculoviruses, or 15 plant cells and plants.

36. Chimeric gene according to claim 35, characterized in that the host organism is a plant cell or a plant which contains it .

37. Chimeric gene according to claim 36, 20 characterized in that the 5' regulatory element comprises regulatory sequences which are promoters in plant cells and plants, and are chosen from promoters which are expressed in plant leaves, constitutive promoters, or light-dependent promoters of bacterial, 25 viral or plant origin.

38. Chimeric gene according to claim 36, characterized in that the 5' regulatory element comprises regulatory sequences which are promoters in 69 plant cells and plants, and are chosen from seed specific promoters.

39. Chimeric gene according to claim 38, characterized in that the promoter is chosen from the 5 promoters for napin, phaseolin, glutenin, zein, helianthinin, albumin and oleosin.

40. Chimeric gene according to one of claims 34 to 39, characterized in that the nucleic acid sequence which encodes an SAT encodes an SAT as defined 10 in claims 2 to 30.

41. Chimeric gene according to one of claims 34 to 39, characterized in that the nucleic acid sequence which encodes an SAT is the nucleic acid sequence according to claim 33. 15 42. Cloning and/or expression vector for transforming a host organism, characterized in that it contains at least one chimeric gene as defined according to one of claims 34 to 41.

43. Method of transforming host organisms, 20 characterized in that at least one nucleic acid sequence according to claim 33, or a chimeric gene according to one of claims 34 to 41, is integrated into the genome of the said host organism.

44. Method according to claim 43, by means 25 of the vector according to claim 42.

45. Method according to either of claims 43 and 44, characterized in that the host organism is chosen from bacteria, for example E. coli, yeasts, in 70 particular of the genera Saccharomyces, Kluyveromyces or Pichia, fungi, in particular Aspergillus, baculoviruses, or plant cells and plants.

46. Method according to claim 45, 5 characterized in that the host organism is a plant cell or a plant which contains it.

47. Method according to claim 46, characterized in that the plant is regenerated from a transformed plant cell. 10 48. Method according to claim 47, characterized in that the host organism is a monocotyledonous plant, in particular chosen from cereals, sugar cane, rice and maize, or a dicotyledonous plant, in particular chosen from 15 tobacco, soybean, rape, cotton, beet and clover.

49. Transformed host organism, characterized in that it comprises at least one nucleic acid sequence according to claim 33, or a chimeric gene according to one of claims 34 to 41. 20 50. Host organism according to claim 49, characterized in that it is obtained by the method according to one of claims 43 to 48.

51. Plant cell, characterized in that it comprises at least one nucleic acid sequence according 25 to claim 33, or a chimeric gene according to one of claims 34 to 41. 71

52. Genetically modified plant, characterized in that it comprises at least one plant cell according to claim 51.

53. Plant according to claim 52, 5 characterized in that the plant is regenerated from a plant cell according to claim 51.

54. Genetically modified plant, characterized in that it is derived from the culture and/or crossing of regenerated plants, according to 10 claim 53.

55. Genetically modified plant according to one of claims 52 to 54, characterized in that it is a monocotyledonous plant, in particular chosen from cereals, sugar cane, rice and maize, or a 15 dicotyledonous plant, in particular chosen from tobacco, soybean, rape, cotton, beet and clover.

56. Genetically modified plant according to one of claims 52 to 55, characterized in that it comprises other genes of interest. 20 57. Genetically modified plant according to claim 56, characterized in that it comprises at least one other gene which modifies the content and quality of the proteins of the said plant, in particular in the leaves and/or seeds. 25 58. Genetically modified plant according to either of claims 56 and 57, characterized in that the gene encodes a protein enriched in sulphur-containing amino acids. 72

59. Seeds of genetically modified plants according to one of claims 52 to 58.