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  • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
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  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
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  • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
  • C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis
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  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance

Definitions

• 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.
• the increase in the nutritional quality of foods derived from legume seeds has been obtained by supplementation with chemically synthesized free methionine.
• 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).
• plants overexpressing a protein rich in methionine and cysteine in their seed 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).
• 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.
• cysteine 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.
• heavy metals for example
• herbicides via glutathione S-transferase activity
• glutathione S-transferase activity which are toxic to the plant, and in the plant's defense mechanisms against microorganisms.
• 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- ph ⁇ sphohomoserine) is derived from aspartate.
• the aspartate is also the precursor in the synthesis of lysine, threonine and isoleucine.
• the diagram indicates the presence of a potential limiting step for the synthesis of methionine, by regulation at the transcriptional level of CGS (cystathionine ⁇ -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]).
• 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 ⁇ ' m (substrate concentration giving half the maximum speed) of the order of 200 ⁇ M to 500 ⁇ M 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 ⁇ M [21].
• 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.
• SAT Serine acetyltransferase
• 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.
• SAT sensitive to cysteine can in particular be a SAT sensitive to cysteine.
• a plant SAT for example a chloroplastic or mitochondrial SAT (SAT2.
• SAT insensitive SAT in the cysteine
• a SAT plant for example a cytoplasmic SAT (SAT-3), or an SAT of mutated bacterial, rendered insensitive cysteine mutaeénippoe J22] and [23] . the contents of which are incorporated here by reference
• the SAT is a SAT of Arabidopsis thaliana [12].
• 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.
• 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.
• 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.
• the non amputated cytoplasmic SAT of its addressing signal is the LOAS 'represented by SEQ ID NO 3
• 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.
• mitochondrial SAT is the LOAS (U22964) represented by SEQ ID NO 4.
• 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.
• the SAT is --incommunée 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.
• 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.
• 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.
• 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
• the SAT can be homologous or heterologous to the transit peptide.
• the fusion protein is the SAT2 or SAT4 protein expressed naturally in the chloroplasts of plant cells.
• 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.
• 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.
• 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.
• 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.
• OTP optimized 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.
• 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.
• nucleic acid sequence coding for a SAT 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.
• host organism 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
• plant cell 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.
• plant 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.
• 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.
• promoters of plant origin promoters histone be mentioned as described in EP 0 507 698. or rice actin promoter (US 5,641, 876).
• 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).
• 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 1 ⁇ TS3 (WO 99/20275).
• 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.
• transcription activators 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.
• 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.
• transformation vectors according to the host organism to be transformed are well known in the art and widely described in the literature.
• 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.
• the vector for transforming plant cells or plants according to the invention is a plasmid.
• 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.
• selectable marker genes include 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.
• 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.
• 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.
• 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.
• 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.
• 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 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].
• 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).
• HPLC high performance liquid chromatography
• OPA orthophthalaldehyde
• 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 ⁇ l, at 25 ° C. After 10 to 15 min of incubation, the reaction is stopped by adding 50 ⁇ l 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 ⁇ l 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 ⁇ -mercaptoethanol) and incubated for 2 min.
• a fraction of this mixture (20 ⁇ l) 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.
• 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.
• the activity of the serine acetyltransferase of the chloroplast of spinach leaves is sensitive to cysteine.
• 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.
• the inhibition constant K ⁇ was estimated to be around 30 (+ 2.2) ⁇ M (variable substrate serine), and 22 ( ⁇ 2 ⁇ M) (variable substrate: acetyl- CoA).
• 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 ⁇ M) using kinetic equations.
• classics [29] Segel, IH (1995), John Wiley and Sons, New York).
• 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.
• the ratio of the activity o-acetylserine (thiol) lyase ( ⁇ ASTL) by serine acetyltransferase activity (SAT) accounts for the large excess of OASTL on the SAT.
• ⁇ ASTL activity o-acetylserine
• SAT serine acetyltransferase activity
• 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.
• 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.
• SAT serine acetyltransferase
• ⁇ ASTL O-acetylserine
• the functional cysteine synthase complex in the cell is represented by the association of two molecular populations.
• 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.
• SAT3 putative cytoplasmic serine acetyltransferase isoform
• 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
• 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]
• the SAT 3 gene (L34076) has a structure without introns.
• 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] .
• 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. .
• Example 3 The procedure of Example 3 is repeated with the following oligonucleotides 3 and 4:
• Oligo 3 5 * GAGAGAGGAT ⁇ TCTTATCG 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).
• SEQ ID NO 2 The structure of the N-terminal (absence of the necessary conditions allowing addressing in an organelle) indicates that this isoform has a cytosolic localization.
• cysteine 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].
• J. lhaliana has at least two cytosolic isolates: SAT3 (example 3) and SAT3 '(or U30298. Example 4).
• the gene corresponding to SAT3 ' has an intron.
• 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 are the following:
• Oligo 5 5 'GAGAGAGGAT ⁇ CCTCCTCC 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 '.
• 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.
• 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].
• 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).
• 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 3 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.
• SEQ ID NO 5 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.
• 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
• 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 ⁇ GACAAGTTGG CATAATTTAT GGCTTGTATA
• Oligo 11 5'TACCTCGTAC CACTCAGAAC TCTAGAAACT CGAGAGAGAG3 '
• 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).
• 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.
• 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
• 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.
• CaMV cauliflower mosaic virus
• 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
• NPTIl kanamycin resistance gene
• the promoter constituting the tobacco mosaic will be replaced by a promoter which allows
• 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 ⁇ -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).
• 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 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
• 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).
• 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.
• 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.
• 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.
• 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 ( ⁇ AS) 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]).
• 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).
• a measure of the content o-acetylserine (free ⁇ AS) was performed.
• the level of OAS in the plant cell average rate of 4 nmol / g fresh matter for the control plants, 6 independent measurements
• 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.
• cysteine 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 (t ⁇ peptide associated with many defense mechanisms of the plant and considered as a reservoir of cysteine) and methionine.
• 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).
• 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 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.
• SMM S-methylmethionine
• 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.
• 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).
• 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) .
• 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.
• 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. ⁇ AS) 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).
• 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.
• Example 12 Analysis of the results for the SAT1 transgenic plants (CDNA U22964 or SATlj, form with transit peptide) and controls.
• the methionine content is multiplied by 2 to 3 times compared to the natural level measured in the control plants.

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