EP1141349A1 - Method for increasing the content in sulphur compounds and particularly in cysteine, methionine and glutathione in plants and plants obtained - Google Patents

Abstract

The invention concerns a method for increasing the production of cysteine, methionine, glutathione and their derivatives in plant cells and plants. Said method consists in over-expression of a serine acetyltransferase (SAT) in the plant cells. The invention also concerns plants containing said plant cells.

Description

 Process for increasing the content of sulfur-containing compounds and in particular cysteine, methionine and glutathione in plants and plants obtained

Methionine is the first essential limiting amino acid in plants, in particular legumes, which are one of the bases of animal nutrition. Cysteine, another sulfur amino acid, is not an essential amino acid, but can be considered as a limiting element for animal nutrition since cysteine is derived, in animals, from methionine. In corn, sulfur amino acids are also limiting amino acids after lysine and tryptophan. In fact, the majority of reserve proteins in the seeds of these plants are poor in these amino acids. The overproduction of methionine and cysteine in the seeds of legumes (soybeans, alfalfa, peas, ...) and corn will therefore have a considerable impact on the nutritional quality of these seeds. Up to now, the increase in the nutritional quality of foods derived from legume seeds has been obtained by supplementation with chemically synthesized free methionine. For example, the average methionine + cysteine content of soybeans and peas is around 20 mg per g of protein. This content must be increased to a value of around 25 mg cysteine + methionine / g of protein to cover the dietary requirements of adult humans, and to a value of around 48 mg of cysteine + methionine / g of proteins to cover those of pigs (De Lumen, BO, Food Technology (1997) 51, 67-70).

The protein characterization techniques enriched in sulfur amino acids and preparation of transgenic plants for the ^expression of such proteins in order to increase the content of sulfur in these plants amino acids, and thus their nutritional value to ^animal feed, thus reduce ^the intake of methionine synthesis, are now well known and described in the literature ([l] orit, AA et al .. Eur J Biochem (1991) 195, 329-334;.. WO 98/20133 ; WO 97/41239; WO 95/31554; WO 94/20828; WO 92/14822). The enrichment of proteins with high sulfur amino acid contents by such an approach remains however limited to the capacity of plant cells and plants to produce said sulfur amino acids necessary for the synthesis of the protein. In fact, plants overexpressing a protein rich in methionine and cysteine in their seed, such as for example lupins expressing albumin 8 S, have a lower methionine, free cysteine and also glutathione level than the control plants ([ 2] Tabe, L. & Droux, M., 4 ^{, h} Workshop on Sulfur Metabolim, in press).

Likewise, peptides rich in sulfur amino acids exhibiting antifungal or antibacterial activity have been identified (WO 97/30082, WO 99/02717, WO 9909184, WO 99/24594, WO 99/53053). The expression of these peptides in plants increases the ability of said plants to resist certain fungal or bacterial attacks. Again, the production of such peptides in plants remains limited to the ability of plant cells and plants to produce the sulfur-containing amines necessary for the synthesis of these peptides. Indeed, the expression of these peptides in the plant cell is at the expense of the glutathione stock, considered as a reservoir for cysteine.

However, it has been found that the limiting parameter of such an approach is indeed linked to this ability to produce methionine or cysteine. It is therefore important to be able to modify in plants this capacity to produce methionine and cysteine in sufficient quantities to allow the production of heterologous proteins with a high content of sulfur-containing amino acids, that is to say to implement a strategy molecular aiming to increase the levels of cysteine and methionine in plants, and more particularly crop plants of agronomic interest.

In plants, the biosynthesis of methionine is carried out from cysteine, this same cysteine being involved in the synthesis of glutathione.

Glutathione is a form of storage of reduced sulfur and represents 60 to 70% of organic sulfur in the cell. Glutathione plays an important role for plants in resistance against oxidative stress and the elimination of toxic compounds. It thus participates in the elimination of xenobiotic compounds: heavy metals (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 defense mechanisms against microorganisms. By increasing the cysteine content of a plant, and consequently its glutathione content, it is then possible to modulate the response of the plant to the various stresses mentioned above.

It therefore exists from cysteine. two distinct pathways, ^one for the preparation of methionine, the other for the preparation of glutathione (Figure 1) and the various enzymes involved are set out below. The SAT (El) and OASTL (E2) activities are at a metabolic crossroads between the assimilation of nitrogen and organic carbon (serine) and inorganic sulfur (reduced sulfur resulting from the sequence of assimilation and reduction of sulphate , gray frame). Cysteine is then incorporated into proteins, but also participates in the synthesis of glutathione and methionine. For this last amino acid, the synthesis of its carbon skeleton (O- phυsphohomoserine) is derived from ^{aspartate. The} aspartate is also the precursor in the synthesis of lysine, threonine and isoleucine. On the other hand, the diagram indicates the presence of a potential limiting step for the synthesis of methionine, by regulation at the transcriptional level of CGS (cystathionine γ-synthase) ([3] Giovanelli J. in Sulfur Nutrition and Sulfur Assimilation in Higher Plants, (1990) pp. 33-48; [4] Chiba Y. & al. (1999), Science. 286, 1371-1374). Methionine is the precursor of SAM (S-adenosylmethionine) involved in most methylation reactions, and of SMM (S-methylmethionine) considered as a form of transport and storage of methionine ([3]).

In plants, the final stages of cysteine synthesis involve the following two enzymes:

E1) Serine acetyltransferase (EC 2.3.1.30) (SAT):

Serine + acetyl-Coenzyme A * - O-acetylserine + Coenzyme A

E2) 0-acetylserine (thiol) lyase (EC 4.2.99.8) (OASTL):

O-acetylserine + sulfide * "- cysteine + acetate The synthesis of methionine from cysteine involves a succession of the following three enzymes:

E3) cystathionine ^ -synthase (EC 4.2.99.9) (CGS): iJ-phosphohomoserine + cysteine ^ ~ cystathionine - _* - Pi

Pi stands for inorganic phosphate. E4) cystathionine / yase (EC 4.4.1.8) (CBL): cystathionine + HoO ^• - homocysteine + pyruvate + NH4 ^"

E5) methionine synthase (EC 2.1.1.14) (MS): homocysteine + 5-methyItetrahydrofolate • "- methionine + tetrahydrofolate

The synthesis of glutathione from cysteine involves a succession of the following two enzymes:

E6) γ-glutamylcysteine synthethase (EC 6.3.2.2) glutamate + L-cysteine + ATP * ~ γ-glutamylcysteine + ADP + Pi

E7) glutathione synthethase (EC 6. 3.2.3) γ-glutamylcysteine + glycine + ATP * ~ glutathione + ADP + Pi

All these enzymes have been characterized and cloned in plants ([5] Lunn, JE & al .. Plant Physiol. (1990) 94, 1345-1352; [6] Rolland, N & al .. Plant Physiol. (1992) 98, 927-935; [7] Droux, M. & al, Arch. Biochem. Biophys. (1992) 295, 379-390; [8] Rolland, N. & al., Arch. Biochem (1993) 300, 213-222; [9] Ruffet. ML & al .. Plant Physiol. (1994) 104, 597 - 604; [10] Ravanel. S. & al .. Arch. Biochem. Biophys. (1995) 316, 572 - 5584; [1 1] Droux, M. & al, Arch. Biochem. Biophys. (1995) 31, 585-595; [12] Ruffet, ML & al., Eur. J. Biochem. (1995) 227, 500 - 509; [13] Ravanel, S. & al., Biochem. J. (1996) 320, 383 - 392; [14] Ravanel, S. & al., Plant Mol. Biol. (1996) 29, 875 - 882; [15] Rolland, N. & al., Eur. J. Biochem. (1996) 236, 272 - 282; [16] Ravanel, S. & al., Biochem. J. (1998) 331, 639- 648; [17] Droux. M & their. J. Biochem. (1998) 255, 235-245; [18] May, ML, Leaver, CJ. Proc. Natl. Acad. Sci. USA (1994) 91, 10059-10063; [19] Ullmann, P. & al., Eur. J. Biochem. (1996) 236, 662-669; [20] Eichel, J. & al., Eur. J. Biochem. (1995) 230, 1053-1058).

It is known that for the synthesis of cysteine. the enzymes E1 and E2 are present in the three compartments of the plant cell, that is to say, the plastids, the cytosol and the mitochondria (5-6, 9, 12). These three enzymes are called El SAT2 and SAT4 for ^the enzyme (putative) chloroplast, SAT1 to the mitochondrial enzyme, SAT3 and SAT3 ^(SAT52) for the cytoplasmic enzyme. These attributions of the locations are based on the analysis of the sequences. For the enzymes of methionine synthesis, the situation is different since the enzymes E3 and E4 are exclusively located in the plasts (10-1 1, 13-14, 16), while the terminal enzyme E5 is in the cytosol (20).

The enzymes associated with the glutathione biosynthetic pathway are localized in both the chloroplast and the cytosol ([21] Hell. R. and Bergmann. L .. Planta (1990) 180, 603-612).

The enzyme E3, from the methionine synthesis pathway, has a λ ^' _m (substrate concentration giving half the maximum speed) of the order of 200 μM to 500 μM for cysteine (10, 16, [ 22] Kreft, BD. & Al. Plant Physiol. (1 994) 104, 1215-1220). The enzyme E6, from the glutathione synthesis pathway, also has a high K _m for cysteine, of the order of 200 μM [21].

It has now been found that the enzyme chlorine seroplastic acetyltransferase

(Figure 2) and to a lesser extent mitochondrial SAT were inhibited by cysteine. unlike the cytoplasmic enzyme (Figure 2). this inhibition constituting the essential limiting factor for the synthesis of cysteine in plant cells, and downstream of methionine and glutathione.

The present invention therefore consists in increasing the level of cysteine and methionine synthesized in the cellular compartments of plant cells, and in particular in the chloroplastic compartment. Increase in cysteine levels. Sulfur precursor of glutathione and methionine and its derivatives, advantageously makes it possible to increase the level of methionine and / or glutathione in plant cells and plants, and subsequently to improve the production of proteins, natural or heterologous, enriched in sulfur amino acids in plant cells and plants, as well as plant tolerance to different stresses regulated by glutathione.

This increase according to the invention is obtained by overexpressing a Serine acetyltransferase (SAT) in plant cells and plants.

The present invention therefore relates to a process for increasing the production of cysteine, glutathione, methionine and their sulfur derivatives, by plant cells and plants, said method comprising overexpressing SAT in plant cells. and plants containing said plant cells.

The overexpressed SAT can be constituted by any SAT, whether it is of plant origin, in particular SAT2 or SAT4, SATl, SAT3, SAT3 ^' (SAT52), or of any other origin, in particular bacterial, in a native or mutated form or deleted from a fragment, and functional in the synthesis of O-acetylserine.

It can in particular be a SAT sensitive to cysteine. such as, for example, a plant SAT, for example a chloroplastic or mitochondrial SAT (SAT2.

SAT4, LOAS), or SAT native ^of bacterial origin ([22] Nakamori et al .. 1998 Appl Microbiol Approximately, 64, 1607- 161 1;... [23] Takagi H. et al, 1999. Febs Lett. 452.

323-327; [24], Mino K. & al., 1999, Biosci. Biotechnol. Biochem., 63, 168-179).

It can also be an insensitive SAT in the cysteine, such as a SAT plant, for example a cytoplasmic SAT (SAT-3), or an SAT ^of mutated bacterial, rendered insensitive cysteine mutaeénèse J22] and [23] . the contents of which are incorporated here by reference), or any mutated and functional plant SAT in the synthesis of O-acetylserine (the carbon precursor for the synthesis of cysteine).

According to a particular embodiment of ^the invention, the SAT is a SAT of Arabidopsis thaliana [12]. According to a first embodiment of the invention, the SAT is overexpressed in the cytoplasm of plant cells. The SAT is either a plant cytoplasmic SAT, in particular SAT 3 (L34076) or SAT3 'or SAT52 (U30298). represented by SEQ ID NO 1 or SEQ ID NO 2, respectively, or an SAT ^of bacterial origin as defined above. SAT overexpressed in the cytoplasm can also be a non-cytoplasmic plant SAT. for example a chloroplastic or mitochondrial SAT. These non SAT cytoplasmic plant are naturally expressed in the cytoplasm in the form of a precursor protein comprising a ^signal targeting to different cell compartment of the cytoplasm wherein the functional mature SAT is released. To overexpress these mature functional SATs in the cytoplasm, they are cut off from their addressing signal. In that case. the SAT protein overexpressed in the cytoplasm is a SAT of a non-cytoplasmic plant amputated from its address signal or signals to different cellular compartments of the cytoplasm.

According to a particular embodiment of ^the invention, the non amputated cytoplasmic SAT of its addressing signal is the LOAS 'represented by SEQ ID NO 3

According to a second embodiment of the invention, the SAT is overexpressed in the mitochondria. The protein is advantageously expressed in the cytoplasm in the form of a signal / S AT peptide fusion protein. the functional mature SAT being released inside the mitochondria. The signal peptide ^{mitochondrial} addressing is advantageously constituted by at least a mitochondrial targeting signal peptide ^of a plant protein mitochondrial localization as the signal peptide subunit / 3-F1 ATPase tobacco [[25] Hemon P . & al. 1990, Plant Mol. Biol. 15, 895-904], or the signal peptide of SAT1 represented by amino acids 1 to 63 on SEQ ID NO 4.

According to a particular embodiment of ^the invention, mitochondrial SAT is the LOAS (U22964) represented by SEQ ID NO 4.

According to a third embodiment of the invention, the SAT is overexpressed in the chloroplasts of plant cells.

The SAT will be expressed in chloroplasts by any appropriate means, in particular by any means known in the art and widely described in ^the prior art.

According to a particular embodiment of the ^"in \ ention, the SAT is --inexprimée in chloroplasts by integration into the chloroplast DNA ^of a chimeric gene comprising a DNA sequence encoding said SAT, under the control ^of regulatory elements in 5 'and 3' functional in chloroplasts. Techniques ^for inserting genes into chloroplasts, such as regulatory elements suitable for the expression of said genes in chloroplasts are well known to those skilled in the art, and in particular described in the following patents and patent applications: US 5,693,507, US 5,451, 513 and WO 97/32977.

According to another particular embodiment of the invention, the SAT is overexpressed in the cytoplasm in the form ^of a fusion protein transit peptide / SAT. the transit peptide having the function of addressing the SAT to which it is fused towards the chloroplasts. functional mature SAT being released ^within the chloroplast after cleavage at the chloroplast membrane.

In that case. SAT SAT can be a chloroplast ^of plant origin. like SAT2 or SAT4, represented by SAQ ID NO 5 or 6 respectively

SAT can also be a plant cytoplasmic SAT or SAT ^of bacterial origin as defined above. In SAT cytoplasmic also imply SAT non cytoplasmic amputated of their ^signal addressed to a different compartment of the cytoplasm as defined above.

Transit peptides, their structures, modes of operation and their use in the construction of chimeric genes for the targeting of a heterologous protein in chloroplasts, as well as chimeric transit peptides comprising several transit peptides are well known from those skilled in the art and widely described in the literature. These include patent applications following: EP 189 707, EP 218 571 and EP 508 909. and the references cited in these patent applications, the content of which is 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 protein is the SAT2 or SAT4 protein expressed naturally in the chloroplasts of plant cells. In the second case, the transit peptide may be a transit peptide ^of a SAT2, shown as amino acids 1 to 32 of SEQ ID 5, or the transit peptide of a SAT4. represented by amino acids 1 to 30 of SEQ ID NO 6, or else a transit peptide of another protein with plastid location, in particular the transit peptides defined below. Protein localization by means plastid protein expressed in the cytoplasm of plant cells in the form ^of a fusion protein transit peptide / protein, the mature protein is localized in the chloroplast after cleavage of the transit peptide.

A plant EPSPS transit peptide is described in particular in patent application EP 218 571, while a plant ssu RuBisCO transit peptide is described in patent application EP 189 707.

According to another embodiment of the invention, the transit peptide also comprises, between the C-terminal part of the transit peptide and the N-terminal part of the SAT, a sequence part of the mature N-terminal part of a protein with plastid localization, this part of sequence generally comprising less than 40 amino acids of the N-terminal part of the mature protein, preferably less than 30 amino acids, more preferably between 15 and 25 amino acids. Such transit peptide comprising a transit peptide fused to a portion of the N-terminal portion ^of a protein is located in plastids is in particular described in patent application EP 1 89 707, particularly for the transit peptide and part N-terminal of plant RuBisCO ssu.

According to another embodiment of the invention, the transit peptide also comprises, between the C-terminal part of the N-terminal part of the mature protein and the N-terminal part of the SAT, a second transit peptide of a vegetable protein with plastid localization. Preferably, this chimeric transit peptide comprising the association of several transit peptides is an optimized transit peptide (OTP) constituted by the fusion of a first transit peptide, with a sequence part of the mature N-terminal part d '' a protein with plastid localization, which is fused with a second transit peptide. Such an optimized transit peptide is described in patent application EP 508 909, more particularly the optimized transit peptide comprising the transit peptide of the ssu RuBisCO of sunflower, fused to a peptide consisting of the 22 amino acids of the N-terminal part of the RuBisCO ssu of mature corn, fused to the transit peptide of the RuBisCO ssu of corn. 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 consists of at least one transit peptide of a natural plant protein with plastid location 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, the term “nucleic acid sequence ^{” means} a nucleotide sequence which may be of DNA or RNA type, preferably of DNA type, in particular double strand, whether it is of natural or synthetic origin, in particular a DNA sequence for which the codons coding for the fusion protein according to the invention will have been optimized as a function of the host organism in which it will be expressed, these optimization methods being well known to those skilled in the art.

Another subject of the invention is the use of a nucleic acid sequence coding for a SAT according to the invention defined above, in particular for cytoplasmic addressing. mitochondrial or chloroplastic, in a process for the transformation of plants, as a coding sequence making it possible to modify the content of cysteine, methionine and derivatives, and glutathione of transformed plants. This sequence can of course also be used in association with other gene (s) marker (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 ′ regulatory elements capable of functioning in a host organism, in particular plant cells or plants, the coding sequence comprising at least a sequence ^of nucleic acid encoding an SAT as defined above.

By host organism is meant any mono or multicellular organism, lower or higher, into which the chimeric gene according to the invention can be introduced. It is ^a question in particular of bacteria, for example E. coli, yeasts, in particular of the genera

Saccharomyces or Kluyve omyces, Pichia. mushrooms, especially Aspergillus. ^a baculovirus, or preferably plant cells and plants.

By "plant cell" is meant according to the invention any cell originating from a plant and which can constitute undifferentiated tissues such as calluses, differentiated tissues such as embryos, parts of plants, plants or seeds.

The term "plant" according to the invention means any differentiated multicellular organism capable of photosynthesis, in particular monocotyledons or dicotyledons, more particularly crop plants intended or not for animal or human food, such as corn, wheat, rapeseed, soy, rice, sugarcane, beet, tobacco, cotton, etc.

The regulatory elements necessary for ^the expression of acid sequence nucleic acid coding for a fusion protein according to the invention are well known to those skilled in the art depending on the host organism. They include in particular promoter sequences, transcription activators, terminator sequences, including start and stop codons. The means and methods for identifying and selecting the regulatory elements are well known to those skilled in the art and widely described in the literature.

The invention relates more particularly to the transformation of plants. As promoter regulatory sequence in plants, any promoter sequence of a gene expressing itself naturally in plants can be used, in particular a promoter expressing in particular in the leaves of plants, such as for example so-called promoters of original origin. bacterial, viral or vegetable or also so-called light-dependent promoters such as that of a gene for the small ribulose-biscarboxylase / oxygenase (RuBisCO) subunit of a plant or any suitable known promoter which can be used. Among the promoters ^of plant origin promoters histone be mentioned as described in EP 0 507 698. or rice actin promoter (US 5,641, 876). Among the promoters of a plant virus gene, mention will be made of that of the cauliflower mosaic (CAMV 19S or 35S). or the circovirus promoter (AU 689 31 1).

It is also possible to use a promoter regulatory sequence specific for particular regions or tissues of plants, and more particularly seed-specific promoters ([26] Datla, R. & al., Biotechnology Ann. Rev. (1997) 3, 269 - 296), in particular the promoters of napine (EP 255 378), of phaseoline. glutenin, zein. heliantinin (WO 92/17580). of ^albumin (WO 98/45460), the oélosine (WO 98/45461). ATS 1 or 1ΑTS3 (WO 99/20275). According to the invention, it is also possible to use, in association with the promoter regulatory sequence, other regulatory sequences which are located between the promoter and the coding sequence, such as transcription activators ("enhancer"), such as for example the translational activator of the tobacco mosaic virus (TMV) described in application WO 87/07644. or the tobacco etch virus (VTE) described by Carrington & Freed.

As a terminating regulatory or polyadenylation sequence. any corresponding sequence of bacterial origin, such as for example the terminator nos dAgrobaclerium tumefaciens, or of plant origin, such as for example a histone terminator as described in application EP 0 633 317, can be used. The present invention relates also a cloning and / or expression vector for the transformation of a host organism containing at least one chimeric gene as defined above. This vector also comprises the above chimeric gene. at least one origin of replication. This vector may comprise a plasmid, a cosmid, a bacteriophage or a virus, transformed by the ^introduction of the chimeric gene according to the invention. Such transformation vectors according to ^the host organism to be transformed are well known in the art and widely described in the literature. For transformation of plant cells or plants, it ^s' will include a virus that can be used for the transformation of developed plants and further containing its own elements for replication and expression. Preferably, the vector for transforming plant cells or plants according to the invention is a plasmid.

For the transformation of host organisms, the chimeric gene according to ^the invention may be employed in combination with a selectable marker gene, either in a single vector, both genes being associated convergently, divergently or collinear, or in two vectors employed simultaneously for the transformation of the host organism. Such marker genes and their use for the transformation of host organisms are well known to those skilled in the art and widely described in the literature. Among the genes coding for selection markers, there may be mentioned the antibiotic resistance genes, the herbicide tolerance genes (bialaphos, glyphosate or isoxazoles), genes coding for easily identifiable enzymes such as the enzyme GUS (or GFP , ^* 'Green Fluorescent Protein "), genes coding for pigments or enzymes regulating the production of pigments in transformed cells. Such selection marker genes are described in particular 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.

The invention also relates to a transforming host organisms method, in particular plant cells by integration of at least a sequence ^of nucleic acid or a chimeric gene as defined above, which transformation may be obtained by any appropriate known means, fully described in the specialized literature and in particular the references cited in the present application, more particularly by the vector according to the invention.

A series of methods involves 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 transfer into the plant a chimeric gene inserted in a Ti plasmid of Agrobacterium tumefaciens or Ri of Agrobacterium rhizogenes. Other methods can be used such as micro-injection or electroporation, or even direct precipitation in PEG. Those skilled in the art will choose the appropriate method depending on the nature of the host organism, in particular the plant cell or the plant.

The present invention also relates to host organisms, in particular plant cells or plants, transformed and containing a chimeric gene defined above. The present invention also relates to plants containing cells transformed, especially plants regenerated from transformed cells. The regeneration is obtained by any suitable process which depends on the nature of the species, as for example described in the references above. For the processes of transformation of plant cells and regeneration of plants, mention will be made in particular of the following patents and patent applications: 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. 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, WO 91/02071 and WO 95/06128.

The present invention also relates to the transformed plants resulting from the culture and / or the crossing of the regenerated plants above, as well as the seeds of transformed plants.

The transformed plants obtainable according to the invention can be of the monocotyledon type such as for example cereals, sugar cane, rice and corn or dicotyledons such as for example tobacco, soybeans, rapeseed, cotton, beet, clover, etc.

The plants transformed according to the invention may contain other genes of interest, conferring on plants new agronomic properties. Among the genes conferring new agronomic properties on transformed plants, there may be mentioned the genes conferring tolerance to certain herbicides, those conferring tolerance to certain insects, those conferring tolerance to certain diseases. Such genes are described in particular in patent applications WO 91/02071 and WO 95/06128. Mention may also be made of the genes modifying the constitution of the modified plants, in particular the content and quality of certain essential fatty acids (EP 666 91 8) or also the content and quality of the proteins, in particular in the leaves and / or seeds of said plants. Mention will in particular be made of the genes coding for proteins enriched in sulfur amino acids ([1]; WO 98/20133; WO 97/41239; WO 95/3 1554; WO 94/20828; WO 92/14822, US 5,939,599 , US 5,912,424). These proteins enriched in sulfur amino acids will also have the function of trapping and storing cysteine and / or excess methionine, making it possible to avoid the possible problems of toxicity linked to an overproduction of these sulfur amino acids by trapping them.

Mention may also be made of genes coding for peptides rich in sulfur amino acids and more particularly in cysteincs, the said peptides also have an antibacterial and / or antifungal activity. Mention will more particularly be made of plant defensins, as well as lytic peptides of any origin, and more particularly the following lytic peptides: androctonine (WO 97/30082 and WO 99/09189), drosomicine (WO 99/02717) , thanatine (WO 99/24594) or heliomicine (WO 99/53053). These other genes of ^interest may be combined with the chimeric gene according to the invention or by conventional crossing two plants each containing one of the genes (the first chimeric gene of the invention and the second gene coding for the protein interest) or by performing the transformation of plant cells ^of a plant containing the gene encoding the protein of interest with the chimeric gene of the invention.

The following examples serve to ^illustrate the invention without, however, seeking to limit its scope.

All the methods or operations described below in these examples are given by way of examples and correspond to a choice made from among the different methods available to achieve the same result. This choice does not affect the quality of the result and therefore, any suitable method can be used by those skilled in the art to achieve the same result. Most of the methods for engineering DNA fragments are described in "Current Protocols in Molecular Biology" 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.SambrookJ982.

The content of all the references cited in the description above and in the examples below is incorporated into the content of the present patent application by reference.

EXAMPLE 1 Demonstration of the Inhibition of the Chloroplastic Serine Acetyltransferase of Pea Leaves (Pisum sativum) by Cysteine

The three subcellular compartments corresponding to the cytosol are prepared from pea leaves (preparation from a subcellular fractionation of pea protoplasts. [12]). mitochondria and chloroplasts [12]. Soluble proteins are extracted therefrom and the serine acetyltransferase activity present in each of the compartments is measured using a technique described [12, 17].

Description of the dosing method:

The activity of the serine acetyltransferase is measured by high performance liquid chromatography (HPLC), by assaying the O-acetylserine formed during the reaction (reaction 1), after derivatization with orthophthalaldehyde (OPA). A defined amount of the protein extract. corresponding to cytosol. and to the various soluble fraction of chloroplasts (stroma) and mitochondria (matrix), is desalted on a PD10 column (Pharmacia) previously equilibrated in 50 mM Hepes-HCl buffer, pH 7.5 and 1 mM EDTA. The enzymatic reaction is carried out in the presence of 50 mM Hepes-HCl, pH 7.5, ImM dithiotreitol, 10 mM L-serine. 2.5 mM acetyl-CoA, in a reaction volume of 100 μl, at 25 ° C. After 10 to 15 min of incubation, the reaction is stopped by adding 50 μl of 20% trichloroacetic acid (W / V). The proteins thus precipitated are then removed by centrifugation for 2 min at 15,000 g. The supernatant, containing the reaction product (OAS), is mixed with 500 μl of a derivatization solution (54 mg of OPA dissolved in 1 ml of absolute ethanol, 9 ml of a borate -NaOH 400 solution mM, pH 9.5 and 0.2 ml of 14 M β-mercaptoethanol) and incubated for 2 min. A fraction of this mixture (20 μl) is injected onto a reverse phase column (AccQ Tag CJS column, 3.9 X 150 mm, Waters) connected to an HPLC system. The buffers used for the elution of the compounds derived from OPA are: Buffer A, 85 mM sodium acetate, pH 4.5, and 6% acetonitile (V / V), pH 4.5; Buffer B. acetonitrile. 60% (VV) in water. O-acetylserine, derived by OPA, is eluted by a continuous linear gradient of buffer B in buffer A from 25 to 70% (V / V), and detected by fluorescence at 455 nm (excitation at 340 nm) . The retention time of O-acetylserine is in our conditions of the order of 6.2 min, and the amount of product formed in the enzymatic tests is quantified from a standard curve produced with O-acetylserine. The enzymatic tests have been optimized in order to respect the optimal pH of the reaction, the linearity as a function of time, and in order to operate in saturating concentrations of substrates.

Effect of cysteine on the serine acetyltransferase activity of pea leaves The incubation (2 min) is carried out in the presence of the protein extract (cytosol, matrix and stroma), and increasing concentrations of L-cysteine (from 0 to 1 mM), before adding saturated concentrations of the substrates of serine acetyltransferase, L-serine (10 mM) and acetyl-CoA (2.5 mM). The enzymatic reaction and the determination of the residual O-acetylserine in the supernatant are carried out as described above. The result of these experiments is shown in the graph in Figure 2 in the appendix.

If one adds the free cysteine (0 to 1 mM, Figure 2) to the various tests, there is a very strong inhibition of serine acetyltransferase activity chloroplast ^(inhibition constant of about 30 microM). Mitochondrial serine acetyltransferase activity is inhibited, but for higher concentrations of cysteine (inhibition constant of the order of 300 μM). On the other hand, the cytosolic serine acetyltransferase activity is insensitive to inhibition by cysteine up to concentrations of the order of 1 mM (FIG. 2). This result therefore proves that only the chloroplastic serine acetyltransferase activity, therefore the enzyme associated with the sulfate assimilation pathway, is inhibited by the final product, L-cysleinc. Table I: Determination of the specific activities and the IC50 values for cysteine for each of the isoforms of serine acetyltransferase.

The concentration of L-cysteine allowing the obtaining of 50% inhibition (IC50) under the standard conditions of the reaction calculated for different enzymatic preparations is shown in Table I. The determinations of the enzymatic activity of the serine acetyltransferase and of the IC50 were carried out in 9 different experiments (stroma), and in 3 for cytosolic extracts and from the mitochondria. Likewise, the activity of the serine acetyltransferase of the chloroplast of spinach leaves is sensitive to cysteine. At the opposite, in Arabidopsis thaliana it appears that only the activity of ^the isoform associated with the cytosolic compartment is controlled by the level of cysteine ([27] Mr. Noji et al., 1998, J. Biol. Chem. 273, 32739-32745; [28] Inoue K. & al. 1999, Eur. J. Biochem. 266, 220-227). For these authors, the activity associated with the chloroplastic compartment is insensitive to cysteine. It would therefore seem that the inhibition of chloroplastic serine acetyltransferase activity by cysteine is a plant-specific phenomenon, but in particular very marked in legumes such as peas.

Study of the mode of inhibition of serine acetyltransferase activity by cvstein The speed of the enzymatic reaction was determined for fixed concentrations of cysteine (0 μM; 10 μM; 20 μM; 40 μM; 60 μM and 100 μM) in varying either the concentration of L-serine or acetyl-CoA. 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 speed of the reaction is modified. The more the concentration of L-cysteine increases, the more the speed of synthesis of O-acetylserine is reduced. For each of the conditions analyzed, the inhibition constant K \ was estimated to be around 30 (+ 2.2) μM (variable substrate serine), and 22 (± 2 μM) (variable substrate: acetyl- CoA). We have been able to show that cysteine is an inhibitor of non-competitive type for serine acetyltransferase activity and moreover of allosteric type (Hill constant of the order of 1.6 ± 0.3 μM) using kinetic equations. classics ([29] Segel, IH (1995), John Wiley and Sons, New York). These results indicate that inhibition of the enzyme chloroplastic takes place at a site different from the active site, and moreover which does not exist on the isoform serine acetyltransferase, present in cystosol.

These inhibition constant values are consistent with the cysteine concentration determined in pea chloroplasts of 40 ± 10 μM (2 nmoles / mg chlorophyll), a value which is calculated for a volume of the stromatic compartment of the order of 35 to 65 μl per mg of chlorophyll.

Dissociation of the bi-enzymatic complex, cysteine synthase, by cysteine

Serine acetyltransferase of the plant cell, such as its bacterial homologous forms an enzymatic complex with o-acetylserine (thiol) lyase, ^the enzyme which catalyzes the condensation of the sulfur with reduced O-acetylserine. This bienzymatic complex is called cysteine synthase. All of the chloroplast serine acetyltransferase exists in a complex form with O-acetylserine (thiol) lyase, while the majority of 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.

Table II: Specific activity of the serine acetyltransferase and O-acetylserine (thiol) lyase activities of the pea leaf cell compartments

The ratio of ^the activity o-acetylserine (thiol) lyase (ΟASTL) by serine acetyltransferase activity (SAT) accounts for the large excess of OASTL on the SAT. In particular in the stroma (chloroplast) where the assimilation and reduction of the sulfate takes place, and in the cytosol, 95% of the OASTL activity is in free form. These conditions are necessary for optimal synthesis of cysteine [14]. The cysteine synthase complex is composed ^of a tctramère serine acetyltransferase and two dimèics of O-acetylserine (thiol) lyase. O-acetylserine, the reaction product of serine acetyltransferase, dissociates this bienzymatic complex, and sulfur tends to stabilize it [14]. These protein-protein interactions within the complex confer new properties on each of the enzymes, in particular the serine acetyltransferase acquires new catalytic properties compared to the free form. Moreover. O-acetylserine (thiol) lyase complex is inactive in the synthesis of cysteine. and only the free form (in excess in the cell) catalyzes the synthesis of cysteine [14].

A chloroplastic fraction (Pisum sativum), previously incubated in the presence of an optimal concentration of cysteine (0J mM), leading to the inhibition of the serine acetyltransferase (see FIG. 2) is then subjected to a gel filtration chromatography allowing the separation molecules according to their molecular mass. Under these conditions the cysteine synthase complex dissociates into tetramers of serine acetyltransferase and dimers of O-acetylserine (thiol) lyase. The serine acetyltransferase chloroplast in its free form is always sensitive to ^inhibition by cysteine. Afiner for this result and confirm that the enzyme inhibition ^is not dependent on the interaction with the OASTL, a serine acetyltransferase has been partially purified from pea chloroplast by chromatography ^of exchange of ions followed by molecular filtration chromatography performed in the presence of O-acetylserine (1 M). a condition which results in the dissociation of the complex.

The fraction thus serine acetyltransferase free from contamination by ^the O-acetylserine (thiol) lyase is incubated in the presence of increasing concentrations of cysteine in the conditions described in Table I and Figure 2. The calculated LC is of the order of 15 +/- 3 micromolar and is comparable to the value obtained previously for the enzyme under the conditions of the chloroplast (see Table I). This last result makes it possible to establish a model for accounting for the inhibition of chloroplastic serine acetyltransferase. In figure 3. the tetrameric form of the serine acetyltransferase (SAT) is represented by the gray circles and the dimer of O-acetylserine (thiol) lyase (ΟASTL) by the black circles. The functional cysteine synthase complex in the cell is represented by ^the association of two molecular populations. In the presence of cysteine, the cysteine synthase complex fixes cysteine which modifies the protein-protein interactions within the cysteine synthase complex. and leads to dissociation into SAT tetramers and OASTL dimers. SAT thus in its free form is also sensitive to cysteine, and we know that this structure tends to form aggregates (outside the cysteine synthase complex) and which result in a loss of its activity [14].

Example 2. Isolation and characterization of a gene coding for a putative cytoplasmic serine acetyltransferase isoform (SAT3) [12] The procedure described on page 502 of Ruffet & al. [12], in particular the chapters described under the titles "Bacterial strain and growth conditions" and "Isolation of A. thaliana serine acetyltransferase cDNA clones by complementation in E. coli". A gene coding for a putative cytosolic serine acetyltransferase (Z34888 or L34076) represented in FIG. 4 (SEQ ID NO 1), was isolated by functional complementation of a strain of Escherichia coli deficient for the serine acetyltransferase activity. Analysis of the primary sequence showed the presence of a strong similarity to the sequence of the bacterial enzyme (56% ^homology and 41% identity).

The primers which were used for the amplification of the nucleotide sequence and its cloning into the vector used for the transformation of tobacco plants are the following: Oligo 1: 5 'GAGAGAGGAT CCTCTTTCCA ATCATAAACC ATGGCAACAT

GCATAGACAC ATGC 3 'Oligo 2: 5' GGCTCACCAG ACTAATACAC TAAATTGTGT TTACCTCGAG

AGAGAG 3 'These primers allow the ^introduction of Bam over restriction sites in 5 ^"(GGATCC) and Sac 3' (GAGCTC).

The N-terminus of the amino acid sequence of the SAT3 isoform does not have the characteristics of addressing peptides in an organelle (mitochondria or chloroplast). This analysis leads to suppose a cytosolic localization for this isoform [12]. The absence of a chloroplastic addressing peptide for this isoform could be confirmed during an import experiment in chloroplasts ([29]

Murillo & al. 1995, Cell. and Mol. Biol. Research 41, 425-433). In contrast, a study using constructs including a portion of the nucleotide sequence and a marker protein (Green Fluorescent Protein, GFP) showed that the presence of the fusion product (5'-SAT3-GFP) in the chloroplast of plants ^of A. processed lhaliana (vegetative stage of the plant) and also in the cytosol (in floral stage). [27]

The SAT 3 gene (L34076) has a structure without introns.

Example 3. Over-expression and purification of SAT3 in Escherichia coli

The protocol defined for the overexpression of the enzyme in E. coli allows the purification of the enzyme in its free form or in complex with the plant O-acetylserine (thiol) lyase, the cysteine synthase complex [14] . With purified proteins, the effect of cysteine on the activity of serine acetyltransferase was analyzed by a spectrophotometric assay based on the consumption of acetyl-CoA during reaction i, as a function of the 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 4200 M " ¹ cm ^-1 ) ([30] Kredich, NM & al., J. Biol. Chem. (1969) 244, 2428-2439). We were able to show that this isoform (SAT3) under its free form or in complex with O-acetylserine (thiol) lyase is insensitive to cysteine. This result allows us to confirm that this cDNA (L34076, Figure 4) codes for a cytosolic serine acetyltransferase, because the amino acid composition of the N-terminal does not have the characteristics of transit peptides, and moreover this serine acetyltransferase is insensitive to cysteine. This last result is similar to the observations obtained for the cytosolic serine acetyltransferase activity of pea leaves (Figure 2 and Table I).

Example 4. Isolation and Characterization of a Gene Encoding a Cytoplasmic Serine Acetyltransferase Isoform (SAT3 ') (U30298)

The procedure of Example 3 is repeated with the following oligonucleotides 3 and 4:

Oligo 3: 5 * GAGAGAGGAT ÇÇTCTTATCG CCGCGTTAAT ATGCCACCGG CCGGAGAACTC C 3 'Oligo 4: 5' GAGCCTTACC AGTCTAATGT AGTATATTTC AACCTCGAGA

GAGAG 3 'A gene coding for an acetyltransferase (U 30298) represented in FIG. 5 is isolated (SEQ ID NO 2). Analysis of the primary sequence shows the presence ^of strong similarity with the sequence of the bacterial enzyme (51.6% homology and 42.6% identity). The structure of the N-terminal (absence of the necessary conditions allowing addressing in an organelle) indicates that this isoform has a cytosolic localization. On the other hand, it is given sensitive to cysteine [27]. This result differs from the data obtained with pea (and spinach) leaves. in the sense that the cysteine regulatory site appears to be confined to the cytosol in A. thaliana. [27]. In addition, it would seem that J. lhaliana has at least two cytosolic isolates: SAT3 (example 3) and SAT3 '(or U30298. Example 4). Unlike the SAT3 gene. the gene corresponding to SAT3 'has an intron.

Example 5. Isolation and Characterization of Genes Coding for a Serine Acetyltransferase (SATl ') Isoform

The procedure described for ^Example 3 is repeated for this example.

A gene coding for a serine acetyltransferase (L78443) represented in FIG. 6 (SEQ ID NO 3) was isolated by functional complementation of a strain of Escherichta coli deficient for the serine acetyltransferase activity. [12] Analysis of the primary sequence shows strong similarities with the sequence of the bacterial enzyme (52.1% homology and 39% identity). The primers which were used for the amplification of the nucleotide sequence and its cloning in the vector used for the transformation of tobacco plants (in bold characters in FIG. 3) are the following:

Oligo 5: 5 'GAGAGAGGAT ÇÇCCTCCTCC TCCTCCTCCT ATGGCTGCGT GCATCGACAC CTG 3'

Oligo 6: 5 'GCTCACCAGC CTAATACATT AAACTTTTTC AGCTCGAGAG

AGAG 3 'These primers allow the instruction of the restriction sites BamHI in 5' (GGATCC) and Sacl in 3 '(GAGCTC). A second gene is obtained which codes for a putative mitochondrial serine acetyltransferase (U22964) represented in FIG. 7 (SEQ ID NO 4) by repeating the same procedure with oligo 7 in replacement of oligo 5 as primer in 5 '.

Oligo 7 °: 5 'GAGAGAGGAT CCGGCCGAGA AAAAAAAAAA ATGTTGCCGG TCACAAGTCG CCG 3'

The N-terminal end of the amino acid sequence of the SAT1 isoform has the characteristics of addressing peptides in an organelle (mitochondria or chloroplast). Mitochondrial localization has recently been confirmed by the construction of a fusion protein including the 5 'portion and the "green fluorescent protein" (5'SAT1-GFP) and by the transformation of Arabidopsis thaliana plants [27]. The gene for SATl '(L78443) or SATl (U22964), like its counterpart (SAT3) does not have an intron.

Example 6. Over-expression and purification of SAT1 in Esche chia coli. Location of this isoform in J. thaliana

The protocol defined for the overexpression of the enzyme in E. coli allows the purification of the enzyme (in its form without transit peptide, SAT L78443) in its free form or in complex with O-acetylserine (thiol ) plant lyase, the cysteine synthase complex [14]. With purified proteins, the effect of cysteine on the activity of serine acetyltransferase was analyzed by a spectrophotometric assay based on the consumption of acetyl-CoA during reaction 1, as a function of the incubation time. (see example 3). The analysis was also carried out by an assay of the reaction product (OAS) by HPLC (see example 1). We have been able to show that this isoform (SAT1 ') in its free form or in complex with O-acetylserine (thiol) lyase is insensitive to cysteine. This last result parallels the observations obtained for the mitochondrial serine acetyltransferase activity of pea leaves (Figure 2 and Table I). The latter being inhibited for non-physiological concentrations of cysteine. With a mitochondria preparation obtained from pea leaves, or from protoplasts from cell cultures, the location of this isoform in the mitochondria could be confirmed.

The mitochondrial fraction devoid of plastid and cytosolic contamination was obtained using the protocol defined for the mitochondria of pea leaves [12]. The molecular mass of the polypeptide revealed by the antibodies directed against the peptide [-TKTLHTRPLLEDLDR-] (see amino acid sequence of SATl) is of the order of 34,000 daltons, a value which is in agreement with the mass of the protein obtained by sequence analysis programs for predicting cleavage sites.

Example 7. Isolation and Characterization of Genes Coding for a Serine Acetyltransferase (SAT2) Isoform

The procedure described for Example 3 is repeated for the present example. A gene coding for a serine acetyltransferase (L78444) represented in FIG. 8 (SEQ ID NO 5) was isolated by functional complementation of a strain of Escherichia coli deficient for the serine acetyltransferase activity. [12] Analysis of the primary sequence showed the presence of strong similarities with the sequence of the bacterial enzyme (49.5% homology and 35.4% identity). The primers which were used for the amplification of the nucleotide sequence and its cloning into the vector used for the transformation of tobacco plants (in bold characters in FIG. 8 are the following:

Oligo 8: 5 'GAGAGAGGAT CCGACAAGTT GGCATAATTT ATGGTGGATC TATCTTCCT 3' Oligo 9: 5'CCTGTGTGAT TGTCGTGTAG TACTCTAGAA

ACTCGAGAGA GAG 3 '

These primers allow the introduction of the 5 ^' BamHI restriction sites (GGATCC) and 3' SacI restriction sites (GAGCTC). The analysis of the N-terminal portion of the sequence has characteristics for addressing the protein in an organelle (mitochondria or chloroplast). Unlike the other isoforms described above, the SAT 2 gene is complex and has several introns. Sequence comparison of the SAT2 with its counterparts ⁱⁿ A. thaliana, plants, and ^other organisms suggest an origin of prokaryotic-type (Figure 10). In addition, analysis of the N-terminal sequence using the chloroP program [http://www.cbs.dtu.dk/services/chlorP/] indicates a high probability for the presence of a transit transit peptide. chloroplastic type. Example 8 Isolation and Characterization of Genes Coding for a Serine Acetyltransferase Isoform (SAT4)

A gene coding for a serine acetyltransferase (SAT4) shown in the figure

9 (SEQ ID NO 6) was isolated by functional complementation of a strain of Escherichia coli deficient in serine acetyltransferase activity. [12] Analysis of the primary sequence showed the presence of strong similarities with the sequence of the bacterial enzyme (44.5% homology and 32% identity)

The primers which were used for the amplification of the nucleotide sequence and its cloning in the vector used for the transformation of tobacco plants are the following:

Oligo 10: 5 'GAGAGAGGAT ÇÇGACAAGTTGG CATAATTTAT GGCTTGTATA

AACGGCGAGA ATCGTGATTT TTCTT 3 '

Oligo 11: 5'TACCTCGTAC CACTCAGAAC TCTAGAAACT CGAGAGAGAG3 '

These primers allow the introduction of Baml I restriction sites in 5 ^"

(GGATCC) and Sacl in 3 '(GAGCTC).

The analysis of the N-terminal portion of the sequence has characteristics for addressing the protein in an organelle (mitochondria or chloroplast). The SAT 4 gene, like that of SAT2, is complex and has several introns. Sequence comparison of the SAT4 with its counterparts ⁱⁿ J. thaliana plants, and other organisms suggest an origin of prokaryotic-type (Figure 10). In addition, analysis of the N-terminal sequence using the ChloroP program [http://www.cbs.dtu.dk/services/chlorP/], indicates a high probability for the presence ^of a transit peptide chloroplastic type. Figure 10 represents the comparison of the sequences, it was carried out using the Clustavv program (Vector NTI software). SAT2 and SAT4 are closer to prokaryotic SATs than are SAT3, SATl and SAT52. In addition, the branch also includes a red alga SAT (AB00848) identified as a protein with a chloroplastic localization and sensitive to cysteine ([32] Toda & al. 1998, Biochim. Biophys. Acta 1403. 72-84) . SAT4 is identified on chromosome 4 (Bac clone F8D20, accession AL031 135).

Example 8. Constructions used for the transformation of tobacco plants - small Havanna variety Expression of the transgene in the leaves

Transformations of tobacco plants are carried out via Agrobacterium tuméfaciens EHA105, containing the vector pBI121 (Clonetch) (Figures 1 1 and 12). SAT3 (or SATl 'OR any SAT insensitive to cysteine)

In order to obtain an expression of the SAT3 (SEQ ID NO 1) of Example 2 in the chloroplast (Figure 11), an extension will be introduced at the 5 'position of the cDNA which will allow addressing in this compartment. For this, the optimized transit peptide described above will be used.

Between the left (BG) and right (BD) borders of the T-DNA is cloned a kanamycin resistance gene (NPTIl) coding for the neomycin phosphotransferase used as a selection marker for tobacco processing. The expression of the NPTI1 gene is dependent on the promoter and the terminator of the nopaline synthase of A. tumefaciens. Downstream, the? -Glucuronidase gene cloned between the unique sites ZtamHl and Sacl, is under the control of the cauliflower mosaic virus (CaMV) 35S promoter and the polyadenylation signal of the nopaline synthase gene from Ti plasmid. The insertion of the OTP-SAT3 construct is carried out between the Xho and Sacl sites of the deleted vector of the / - -glucuronidase gene (FIG. 11) SAT1, SAT3, SAT3'- SAT2, SAT4 or all SATs

In order to obtain SAT expression in all subcellular compartments (cytosol, mitochondrion or chloroplast). the transgene will be introduced into the appropriate vector described in FIG. 12.

Between the left (BG) and right (BD) borders of the T-DNA is cloned a kanamycin resistance gene (NPTIl) coding for the neomycin phosphotransferase used as a selection marker for tobacco processing. The expression of the NPTI1 gene is dependent on the promoter and the terminator of the nopaline synthase of

A. tumefaciens. Downstream, the / 3-glucuronidase gene cloned between the unique sites BamHI and SacI, is under the control of the promoter 35S of the driver's mosaic virus (CaMV) and the polyadenylation signal of the nopaline synthase gene of the Ti plasmid. The insertion of the gene coding for a SAT takes place between the BamHI and SacI sites of the deleted vector of the β-glucuronidase gene (FIG. 12). Expression of transgenes in seeds A construction similar to that presented in FIGS. 1 1 or 12 is carried out in order to obtain a specific expression of the transgene in seeds. This strategy could be important since seeds make up the main intake for animal feed. For this, the promoter constituting the tobacco mosaic will be replaced by a promoter which allows a specific expression of trαnsgene during the establishment of the reserves of the seed.

Example 9. Tobacco processing

Dice young leaves of tobacco plants (3 to 4 weeks old) whose surface is sterilized with a 10% bleach solution (V / V) for 10 min then rinsed with water sterile, are cut with a cookie cutter (30 discs per construction). 20 ml of a 48-hour culture of Agrobacterium tumefaciens EHA105 (containing the vector pBI121 modified according to the invention) are centrifuged and then resuspended in 4 ml of a 1 MM MgSO 4 solution. The leaf discs are incubated for a few minutes with the agrobacteria solution and then transferred to MS medium (Sigma M-5519) supplemented with 0.05 mg / 1 of α-naphthalene acetic acid (NAA, Sigma). 2 mg / 1 of 6-benzylaminopurine (BAP) and 7 mg / 1 of phytoagar, for 2 to 3 days. The leaf discs are then transferred to an identical medium to which are added 350 mg / l of cefotaxine (bacteriostatic) and 75 mg / l of kanamycin (selection agent). After 2 weeks, the discs on which calluses have developed as well as young shoots are transplanted onto an identical medium in order to accelerate the growth of the shoots. A week later, the green shoots are excised and transferred to the same hormone-free medium, in order to allow the development of the roots, this for about 2 weeks, at the end of which the young plants are transferred to the ground and cultivated in the greenhouse.

Example 10. Analysis of the results for the transgenic plants SAT3 and SATl '(L78443) (truncated form of SATl U22964) and controls.

The impact of the expression of SAT3, SATl ', OTP-SAT3 in the leaves or in the seeds of tobacco plants is analyzed with regard to the content of sulfur compounds, cysteine and methionine (and derivatives such as S-methylmethionine or SMM) and glutathione. Cysteine and glutathione are demonstrated by the Fahey method ([33] Fahey, RC and Newton, GL, Methods 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 determination of free methionine and SMM is carried out by the methods of determination of free amino acids after extraction and derivation by orthophthaldehyde and separation by HPLC ([34] Brunet, P. & al., J. Chrom. (1988 ) 455, 173-182). The measurement of the activity of the serine acetyltransferase is carried out as described in the assay methodology for the O-acetylserine formed, by the HPLC technique, or by the coupling method in the presence of an excess of O-acetylserine. (thiol) lyase [12], [14]. The activity of the SAT transgene in transformed plants (i.e. in vivo) is revealed by the determination of the O-acetylserine produced during the activity of the enzyme and temporarily accumulated in the cell.

The determination of 1 ^" O-acetylserine in plant extracts follows the following protocol.

After grinding the tobacco leaves into 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 around 10 min, the debris is removed by centrifugation for 15 min at 15,000 g. A fraction of the supernatant obtained, containing the free amino acids is derivatized for 1 min at 25 ° C in the presence of a solution ^of ortho-phthalaldehyde (54 mg solution of ortho-phthalaldehyde, 10% methanol, 90% sodium borate 400 mM, pH 9.5, and 0.2 ml of /? - mercaptoethanol). The OPA-amino acid derivatives are then 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: sodium acetate, 85 mM, pH 4.5 supplemented with final 6% acetonitirile; buffer B: 60% acetonitrile in water. The 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; 9 min, 80% B in A; 10 min, 100% B; 12 min, 100% B. At the column outlet, the fluorescence emitted by the derivatives is measured at 455 nm after excitation at 340 nm (fluorimeter SFM25, Kontron).

The retention time of O-acetylserine under our experimental conditions is 9.5 min. The identity of the peak corresponding to O-acetylserine is confirmed by co-elution with a known quantity of the pure product. In addition, a second check was carried out to confirm the position of O-acetylserine in the various analyzes. The samples, before incubation with the OPA, are previously treated with final 0.2 M NaOH. Under these conditions, the acetate function in the OH position of the serine is transferred to the amine function and thus allowing the formation of .V-acetylserine. The latter compound is no longer detected under our experimental conditions and therefore leads to the disappearance of the peak corresponding initially to O-acetylserine.

The plants transformed with the SAT transgene were previously selected on kanamycin and led to seeds. Control plants (PBI, three independent lines containing the transformation vector and a GUS cassette) are treated identically. Plant analyzes include: 1; Demonstration of ^the transgene insertion in the genome by PCR using the primers 5 ^{'and 3'} corresponding to the SAT used for transformation; 2, as evidenced by an analysis of the messenger messengers using probes corresponding to the SAT transgenes used for the transformation of plants according to known techniques; 3, demonstration of the enzymatic activity associated with the SAT protein according to the methods described in the literature ([14]) and localization of the transgene; 4. dosage of the SAT reaction product, i.e. O-acetylserine (ΟAS) in transformed plants; 5, determination of cysteine and its direct derivatives, glutathione and methionine (and its methylated derivatives); 5, analysis of the total amino acid composition of plants and seeds associated with each of the transgenes obtained (free amino acids and bound to proteins) according to traditional techniques; 6; analysis of the impact of over-e xpression of SAT activity in the plant cell on the content of enzymatic activity associated with the sulfur assimilation sequence (sulfate transporters, ATP-sulfurylase, APS reductase, sulfite reductase and in particular O-acetylserine (thiol) lyase, the enzyme directly associated with SAT activity for the synthesis of cysteine ([14]). In addition, the enzymes associated with the synthesis sequence of methionine and glutathione are analyzed in order to account for the impact of the cysteine content on the metabolism associated with the synthesis of glutathione and methionine. Expression of the Arabidopsis thaliana serine acetyltransferase gene in tobacco leads to an increase in the cysteine level, the glutathione level and the methionine level in the tissues of transformed plants compared to the control plants. In general, this increase in the content of free sulfur compounds is associated with the expression of the transgene in the plant cell (FIG. 13). The measurement is made in the leaves of 3 different plants of each homozygous line. The SAT activity is measured by its capacity to promote the synthesis of cysteine according to the protocol described above ([14]).

The expression of the transgene under the control of the constitutive promoter CaMV leads to increasing the capacity (the maximum potential enzymatic activity measured in vitro) of the SAT by a factor 2 to 8 compared to the level measured in the control plants (transformed plants with an empty vector). To account for ^the actual activity of the SAT transgene, a measure of the content o-acetylserine (free ΟAS) was performed. Thus, the level of OAS in the plant cell (average rate of 4 nmol / g fresh matter for the control plants, 6 independent measurements) could be multiplied by a factor of 2 to 10 times in the transformed plants (2 independent measurements ). Thus, associated with the net increase in the capacity of the enzymatic activity of SAT is associated an increase in the intracellular free OAS which results from the activity of the transgene in vivo, and with an increase in the content of free cysteine in most SAT transgenes compared to control plants (Figure 14). The cysteine content in control plants (PBI) and in T2 tobacco plants transformed with an SAT (SAT1 'and SAT3 lines) are determined as monobromobimane derivatives by HPLC for 3 plants per line ([33]). The cysteine content of transgenic lines is increased from 2 to 10 times compared to control plants (PBI).

The free cysteine content in most transgenic plants expressing SAT is significantly 2 to 10 times higher than the natural level measured in PBI control plants (worth 5 nmol / g fresh matter, average calculated on three independent lines and each containing 5 plants). This impact of the expression of SAT is observed from the TI generation. By cons no correlation n ^'could be found between the content cysteine (and also free OAS, and the activity measured SAT transgenes by in vitro against a positive and significant correlation could be measured between the content. in cellular OAS and the cysteine level of the cell (Figure 15). Compared to control plants, a 3 to 10-fold increase in the level of free O-acetylserine in vivo linked to transgene activity, results in a increase 3 to 8 times the cysteine level in plants. the ^analysis was performed on fully developed leaves (about 2 months) of plants homozygous for the transgene. Control plants are transformed plants with empty constructs (PBI). An increase in the content of free cellular OAS linked to the activity of the transgene SAT in the transformed plants is positively correlated with an increase in the content of cysteine. Thus, an average increase of 6 times in the free OAS level is associated with a 6-fold increase in the cysteine level. The slope associated with the distribution of the points is 1.06 +/- 0.09 (regression coefficient of 0.67). It indicates that for every ^molecule OAS accumulated mole of cysteine is synthesized. The value of the slope and the ^absence tray observed in our experimental conditions indicate that sulfide formation (sulfate assimilation and reduction in sulfide) is not a limiting sequence and the SAT activity appears to be the limiting factor in the cell for the formation of cysteine (Figure 1)

The sub-cellular localization of the SAT1 '(truncated form of SAT1) and SAT3 transgenes in the transformed tobacco plants could be clarified by preparing the chloroplast fraction of the transformed plants having the highest enzymatic activity compared to the plants PBI (controls). The activity associated with the chloroplast compartment is compared with that measured in the total extract (Figure 16). The serine acetyltransferase activity values correspond to 3 lines for PBI (5 plants per line), 5 lines for SAT1 'and SAT3 represented by 5 plants each. The columns in gray correspond to activities measured in ^total realized extracted from each of the lines, and black columns represent the average of the measured activity in each chloroplast preparations.

These results definitively establish that SAT3 is an isoform of serine acetyltransferase localized in the cytosol of the plant cell, and that the truncated form of SATl (absence of transit peptide) is also localized in the cytosolic compartment. With regard to SAT3, these results confirm our interpretations derived from the analysis of the protein sequence [12].

A direct consequence of the increased level of cellular cysteine results in increased synthesis of glutathione and methionine (see Figure 1). The fate of cysteine is multiple and apart from its incorporation at the protein level, its participation in the synthesis of multiple compounds such as vitamins (biotin, thiamine, ... and other sulfur derivatives of the cell), cysteine also participates to the synthesis uu glutathione (tπpeptide associated with many defense mechanisms of the plant and considered as a reservoir of cysteine) and methionine. In fact, in plants transformed with the SAT transgene the level of glutathione is directly correlated to that of cysteine and results in an increase of 2 to 7 times the natural rate measured in controlled plants (PBI) (Figure 17). The correlation coefficient calculated for the distribution of points is 0.92. A 4-fold increase in the content of Cysteine in transgenic tobacco plants overexpressing SAT results in a 3-4 fold increase in the level of glutathione. The analysis was carried out with fully developed leaves (approximately 2 months) of plants homozygous for the transgene. Control plants are transformed plants with empty constructs. This result indicates that cysteine is the limiting factor for the synthesis of glutathione in the plant cell. Therefore, indirectly any modification of the level of serine acetyltransferase in the cell will result, via the increase in the level of cysteine, an increase in the content in intra-cellular glutathione. This result implies that the transgenic plants obtained have acquired stress resistance properties compared to the control plants (PBI). This aspect has been observed recently ([34], Blaszczyl A. & al., 1999, The Plant Journal 20, 237-243). In addition, the content cysteine and glutathione considered a reservoir leads to increased availability in the synthesis of polypeptides rich in cysteine (for example for resistance to plant pathogenic attacks) and cysteine rich in methionine ^(for animal feed)

The increase in cysteine in the plant cell also leads to an increase in the relative methionine content (Figure 18). On the other hand, contrary to the results observed for glutathione, the curve presents a plateau which seems to indicate the existence of another control site which would limit the synthesis of methionine. In addition, homocysteine, derived from the transsulfurization pathway, and the sulfur precursor at the synthesis of cysteine does not seem to accumulate. This observation therefore indicates that the folate pool of the plant cell, essential for methylation and the formation of methionine, is not a limiting factor. This limitation would therefore be located downstream of the cysteine and upstream of the homocysteine. It relates to the synthesis of the carbon precursor for the synthesis of methionine which is derived from aspartate (() - phosphohomoserine and / or cystathionine). The level of aspartokinase (the first enzyme in the aspartate pathway for the synthesis of Lysine, threonine and methionine) is controlled by several effectors such as threonine, and S-adenosylmethionine (SAM) resulting from the synthesis of methionine ([3]). Cystathionine γ-synthasc (see Figure 1) is directly regulated at the transcriptional level [3] and more precisely methionine or one of its derivatives controls the stability of its messenger [4]). The maximum shelf obtained under our experimental conditions is about 39 +/- 7 nmol / g fresh material methionine which corresponds to an increase of the average natural rate ^of about 6 +/- 2 per g material namole fresh (PBI control). The maximum value obtained for methionine requires an increase in the cysteine content of the cell from 4 to 5 times its maximum level. The regression coefficient is 0.50.

In addition, the increase in methionine in cells leads to multiply the level of S-methylmethionine (SMM) from 2 to 10 times depending on the plants. The SMM derives directly from methionine methylathion in the presence of S-adenosylmethionine. This compound is important for the cell and is a form of transport of methyl groups (of methionine) in the plant. Indeed, in the presence of a molecule ^of homocysteine (a sulfur precursor for the synthesis of methionine and cysteine derived), the MMS allows the synthesis of two molecules of methionine ([3], [35], & Bourgis al., 1999, Plant Cell 1 1 J 485-1497). It could therefore have a vital role in the synthesis of reserve proteins in the seed. In addition, SMM is the direct precursor for the synthesis of a compound such as 3-dimethylsulfoniopropionate involved in the resistance of plants to salt stress ([36], Hanson AD & al., 1994, Plant PhysiolJ 05, 103-1 10) . Such an approach has multiple consequences, in particular for increasing the potential of plants on soils rich in salts.

Evidence for a regulatory role of the sulfate assimilation pathway in vivo.

The serine acetyltransferase is considered a limiting factor for ^the sulfur assimilation and synthesis of cysteine. Its role in bacteria is important since the reaction product, (O-acetylserine. ΟAS) or its derivative (N-acetylserine) is the effector which modulates the expression of the genes of the sulfur assimilation sequence as : 1. transport of sulfate. 2, ATP sulfurylase. 3. APS kinase. And 4, PAPS reductase ([37], Kredich NM, 1987, in Escherichia coli and Salmonella typhimurium: cellular and molecular biology, pp. 419-428). In plants, a role of OAS in the modulation of the expression of several genes has been demonstrated and concerns the sulfate transporters, ([38], Smith FW & al. 1997, The Plant Journal 12, 875-884; [39], Hawkesford MJ & al. 1995, Z. Pfanzenernârh. Bodenk. 158, 55-57; [40], Clarkson DT & al. 1999, Plant Physiol. Biochem. 37, 283-290), ATP sulfurylase ([39-40]) and APS reductase ([41 j. Neuenschwander U. & al. 1991, Plant Physiol., 97, 253-258). The role of serine acetyltransferase activity in gene modulation has been postulated according to the kinetic characteristics of complex cysteine synthase (bi-enzyme complex composed of serine acetyltransferase and O-acetylserine (thiol) lyase) ([ 41], Droux & al. In Sulfur and Nutrition in Plants, in Press), and led to describe a model to account for the mechanism of gene regulation. The role of OAS is also decisive in the regulation of gene expression during seed formation ([42], Kim H. & al. 1999, Planta 209, 282-289).

In transgenic plants which overexpress a SAT in the cytosol, a transient increase in OAS could be demonstrated (increase from 2 to 10 times its natural level, see Figure 15). At the same time, in most transgenic plants, an increase in OASTL activity was measured (Figure 19). This increase of 2 to 5 times compared to the measured activity in PBI controls concerns only ^the activity associated with the chloroplast. In addition, in a Western Blot. the signal observed is greater in most transgenic lines (Figure 20) indicating that the 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 the genes of the sulfate assimilation sequence, in particular for the chloroplastic OASTL.

For FIG. 20, the equivalent amount of protein (50 mg) is subjected to an SDS-PAGE (12%) and after separation of the proteins, these are transferred to a nitrocellulose membrane. The presence of the OASTL is revealed by incubation with antibodies against OASTL chloroplast of ^leaf spinach ([7]). The overexpression of SAT in the plant cell therefore leads to increasing the capacity to synthesize cysteine in the chloroplast. It is therefore safe to assume that the expression of the genes coding for the enzymes of the sulfate assimilation and reduction pathway (sulfate transporter, ATP sulfurylase, APS reductase, sulfite reductase) is also modulated like OASTL (and references [38-41]). The increase in intracellular OAS content (which derives from the activity of

SAT) signals an artificial state of sulfur stress (absence of sufficient reduced sulfur) to the cell (in transformed plants) which leads to inducing the enzymes of the sulfate assimilation pathway.

Impact of the increase in cysteine in the cell on the general content of amino acids. This increase in sulfur compounds is accompanied by an increase in the content of essential amino acids such as threonine, isoleucine, and lysine (their content is multiplied by 2 on average). On the other hand, the glutamate level is halved like that of aspartate. This last observation is directly related to ^the increase in the content THR. LYS and ILE. All increases in amino acids are correlated with an increase in the activity of serine acetyltransferase (SAT3, or SATl ') in the cytosol. In addition, the increase in these sulfur compounds leads to improving the N / S nutritional ratio of the plant (based on free amino acids). This resulted in a decrease of the relative ratio due to ^the enrichment of total sulfur compound (cysteine. Methionine, and glutathione SMM). This factor is important because it conditions the content of the seeds in polypeptides and leads to an enrichment (or a depletion if the N / S ratio is too high) of the reserve proteins rich in sulfur amino acids to the detriment of the polypeptides poor in these compounds. .

Example 11 Analysis of OTP-SAT3 (OTP-SAT1 ') Transgenic Plants

Analysis of transformants at the TO stage of transgenic plants expressing a SAT insensitive to cysteine (here as example SAT3 or SATl ", truncated form of SATl U22964), in the leaves or in the seeds (under the control of a specific seed promoter), reveals an increase in the content of free cysteine, but also of glutathione (2.6 times the natural rate), and in methionine. Plants expressing these same isoforms in the cytosol under the control of a specific seed promoter reveal a level of sulfur compound higher than the control plants.

Example 12 Analysis of the results for the SAT1 transgenic plants (CDNA U22964 or SATlj, form with transit peptide) and controls.

The impact of expression of serine acetyltransferase in the mitochondria was analyzed by transforming the plants with the construct (Figure 12) containing the entire sequence of SAT1. Analysis of the plants at the TO stage makes it possible to highlight an increase in free cysteine in the cell (FIG. 21). The analysis is carried out on the leaves formed before the appearance of the flower stalk. The fourteen lines show a 2- to 6-fold increase in cysteine levels compared to the control plant (PBI). The increase in cysteine is accompanied by a general effect on the content of sulfur compounds with a multiplication by 4 of the glutathione content in the cell (Figure 22). In contrast to an expression of SAT in the cytosolic compartment, the general pattern of the distribution of values in the different lines presents a plateau which would indicate a limitation in the synthesis of glutathione. This limitation may concern the level of glutamate and / or glycine, or a control of glutathione on its own synthesis (retro-inhibition of one of the enzymes participating in the synthesis of glutathione, enzyme E6 and / or E7 see Figure 1) .

Likewise, the methionine content is multiplied by 2 to 3 times compared to the natural level measured in the control plants.

Claims

claims

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

2. Method according to claim 1, characterized in that the SAT overexpressed in plant cells is a SAT sensitive to cysteine.

3.

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 overexpressed in plant cells is a SAT insensitive to cysteine.

5. Method according to claim 4, characterized in that the SAT is a plant SAT or a SAT of bacterial or mutated plant origin, made insensitive to cysteine by mutagenesis.

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 a SAT of bacterial origin.

8. Method according to claim 6, characterized in that the SAT is a

Plant cytoplasmic SAT, in particular Arabidopsis thaliana.

9. Method according to claim 8, characterized in that the SAT is SAT 3, represented by SEQ ID NO 1.

10. The method of claim 6, characterized in that the SAT is a SAT of non-cytoplasmic plant amputated from its or its addressing signals to cellular compartments different from the cytoplsame.

1 1. Method according to claim 10, characterized in that the SAT is SAT 1 'represented by SEQ ID NO 2.

12. Method according to one of claims 1 to 5, characterized in that the SAT is overexpressed in the mitochondria.

13. The method of claim 12, characterized in that the SAT is overexpressed in the cytoplasm in the form of a signal peptide / S AT fusion protein, the mature functional SAT being released inside the mitochondria.

14. Method according to claim 13, characterized in that the mitochondrial addressing signal peptide consists of at least one signal peptide of a natural plant protein with mitochodrial location, such as for example the small signal of SAT1 represented by acids amino 1 to 63 on SEQ ID NO 3 ..

15. The method of claim 13, characterized in that the SAT is a mitochondrial SAT of plant origin, in particular of Arabidopsis thaliana.

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

17. The method of claim 6, characterized in that the SAT is overexpressed in the chloroplasts of plant cells.

18. The method of claim 17, characterized in that the SAT is overexpressed in chloroplasts by integration into the chloroplast DNA of plant cells of a chimeric gene comprising a DNA sequence coding for said SAT, under the control of regulatory elements in 5 'and 3' functional in chloroplasts.

19. The method of claim 17, 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 the chloroplasts.

20. The method of claim 19, characterized in that the SAT is homologous to the transit peptide.

21. Method according to claim 20, characterized in that the SAT is a

SAT chloroplastic of plant origin, in particular Arabidopsis thaliana.

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

23. The method of claim 19, characterized in that the SAT is heterologous to the transit peptide.

24. The method of claim 13, characterized in that the SAT is a cytoplasmic SAT of plant origin or a SAT of bacterial origin, as defined in one of claims 3 to 5 or 9 to 1 1.

25. Method according ^to one of Claims 23 or 24, characterized in that the transit peptide is a transit peptide ^of another protein is located in plastids.

26. The method of claim 25. characterized in that the transit peptide consists of the transit peptide of a plant EPSPS or the transit peptide of a plant RuBisCO ssu.

27. Method according to one of claims 25 or 26, characterized in that the transit peptide comprises a transit peptide of a plant protein with plastid location and a sequence part of the mature N-terminal part of a protein with plastid localization between the C-terminal part of the transit peptide and the N-terminal part of the SAT.

28. The method of claim 27. characterized in that the sequence part generally comprises less than 40 amino acids of the N-terminal part of the mature protein, preferably less than 30 amino acids, more preferably between 15 and 25 amino acids .

29. Method according to one of claims 27 or 28, characterized in that the transit peptide comprises between the C-terminal part of the N-terminal part of the mature protein and the N-terminal portion of the SAT a second transit peptide ^of a plant protein is located in plastids.

30. The method of claim 29, characterized in that the transit peptide is an optimized transit peptide (OTP) constituted by the fusion of a first transit peptide, with a part of the sequence of the mature N-terminal part d '' a protein with plastid localization, which is fused with a second transit peptide.

31. Transit peptide / SAT fusion protein, characterized in that the SAT is heterologous to the transit peptide.

32. A fusion protein according to claim 31, as defined in claims 24 to 30.

33. Nucleic acid sequence coding for a transit peptide / SAT fusion protein according to one of claims 31 or 32.

34. Chimeric gene comprising a coding sequence as well as heterologous 5 'and 3' regulatory elements capable of functioning in a host organism, characterized in that the coding sequence comprises at least one nucleic acid sequence coding for a SAT.

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 or Kluyveromyces, Pichia, fungi, in particular Aspergillus, baculovirus, or plant cells and plants.

36. A chimeric gene according to claim 35, characterized in that the host organism is a plant cell or a plant containing it.

37. A gene construct according to claim 36, characterized in that ^the regulating member 5 ^'promoter comprises the regulatory sequences in plant cells and plants, selected from the promoter which is expressed in plant leaves, constitutive promoters , or dependent light promoters of bacterial, viral or plant origin

38. Chimeric gene according to claim 36, characterized in that the 5 ′ regulatory element comprises the promoter regulatory sequences in plant cells and plants, chosen from promoters specific for seeds.

39. Chimeric gene according to claim 38, characterized in that the promoter is chosen from the promoters of napine, of phaseoline. glutenin, zein, heliantinin, albumin or oleosin.

40. Chimeric gene according to one of claims 34 to 39, characterized in that the nucleic acid sequence coding for a SAT codes for a SAT as defined in claims 2 to 30.

41. Chimeric gene according to one of claims 34 to 39, characterized in that the nucleic acid sequence coding for a SAT is the acid sequence nucleic acid according to claim 33.

42. Cloning and / or expression vector for the transformation of a host organism characterized in that it contains at least one chimeric gene as defined according to one of claims 34 to 41.

43. A method of transforming host organisms, 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 said host organism.

44. The method of claim 43, using the vector of claim 42.

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

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

47. The method of claim 46, characterized in that the plant is regenerated from a transformed plant cell.

48. The method of claim 47, characterized in that the host organism is a monocotyledonous plant, in particular chosen from cereals, sugar cane, rice and corn, or a dicotyledonous plant, in particular chosen from tobacco , soybeans, rapeseed, cotton, beets 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.

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. A plant cell, characterized in that it comprises at least one nucleic acid sequence according to Claim 33 or a chimeric gene according ^to one of claims 34-41.

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

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

54. Genetically modified plant, characterized in that it comes from the culture and / or crossing of regenerated plants according to 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 corn, or a dicotyledonous plant, in particular chosen from tobacco, soybeans, rapeseed, cotton, beets and clover.

56. Genetically modified plant according to one of claims 52 to 55, characterized in that it comprises other genes of interest.

57. Genetically modified plant according to claim 56, characterized in that it comprises at least one other gene modifying the content and the quality of the proteins of said plant, in particular in the leaves and / or seeds.

58. Genetically modified plant according to one of claims 56 or 57, characterized in that the gene codes for a protein enriched in sulfur amino acids.

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