FR2829904A1 - Reducing nitrate accumulation in plants, useful for preparing edible plants with reduced nitrate levels, comprises inhibiting the chloroplast chloride channel - Google Patents

Abstract

Reducing (M1) accumulation of nitrate in a plant by inhibiting the chloroplast chloride channel CLC-e, is new. Independent claims are also included for: (1) producing (M2) a plant that under-accumulates nitrate; (2) an expression cassette (EC) containing a polynucleotide (I) comprising at least part of the clc-e gene, or its complement, or sequences with at least 70% identity; (3) a vector containing the EC; (4) a plant cell transformed with the EC; and (5) plants that under-accumulate nitrate, produced by (M2).

Description

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 The present invention relates to the improvement of plants, and more particularly to the production of plants that accumulate nitrate.

 Nitrate is essential for the plant that uses it as a source of nitrogen to synthesize essential amino acids. Plant roots absorb nitrate naturally present in the soil or in the form of fertilizer. Nitrate once absorbed can be transported through the xylem to the aerial parts of the plant.

 Schematically, the incorporation of nitrogen into the amino acid molecules from the nitrate ion involves three main steps. A first step catalyzed by nitrate reductase makes it possible to reduce the nitrate to nitrite. A second step is catalyzed by nitrite reductase which reduces the nitrite ion to ammonium ion. Finally, the ammonium ion will react with glutamic acid to form glutamine, which participates in the biosynthesis of amino acids.

 These reactions occur mainly in the aerial parts of the plant, during the light phase of photosynthesis which provides the necessary electrons for the reduction reactions. The absorption and assimilation of nitrate varies according to the length and intensity of the daytime period since these steps directly depend on the efficiency of photosynthesis.

 The nitrate that is not used is stored in subcellular compartments, such as, for example, the vacuole.

 From a dietary point of view, a high nitrate content in plants whose leaves, roots or bulbs are eaten, especially when these fruits or vegetables are eaten raw, is not recommended. Studies have shown that a significant ingestion of nitrates can increase the methemoglobinemia (or blue disease) of the infant and promote the appearance of certain cancers. In fact, the nitrates ingested are transformed into nitrites in the digestive tract by the bacteria present. In the middle

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 acid, for example in the stomach, nitrites can react with amines or amides to form carcinogenic nitrosamines.

 Given the potential risks associated with the overconsumption of nitrates, standards have been set, for example for salads, regulatory nitrate levels are 2500 ppm in summer and 4500 ppm in winter. These nitrate limit rates are quite regularly exceeded, especially in winter or in countries with a low level of sunshine.

 To overcome this excessive accumulation of nitrates, it is possible to play on the exogenous supply of nitrates by reducing the amounts of fertilizer, by optimizing the periods of amendment, by improving the conversion of nitrate in the soil.

 On the other hand, it is possible to reduce the nitrate content in plants by influencing the efficiency of the absorption, the conversion of nitrate in the plant or by selecting varieties naturally under-accumulating nitrate. For example, in order to decrease the nitrate contents, it has been proposed to modify the activity of the enzymes involved in the nitrate assimilation pathway, such as nitrate reductase.

 Another way of decreasing nitrate accumulation is to convert the nitrate into the plant after harvest. For example, Application EP 0577222 proposes a method for rapidly and significantly decreasing the nitrate content in plants by a CO 2 treatment.

 The present invention now provides a novel approach for obtaining plants that accumulate nitrate. The inventors have now found that it is possible to modulate the accumulation of nitrate in plants by acting on anionic channels, and in particular chlorine channels, the chloroplast membrane. The present invention therefore proposes a novel approach for obtaining plants that accumulate nitrate.

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 By ion channel is meant any protein having at least one transmembrane region allowing the passage of ions. Chlorine channels, also called chloride channels, or CLC channels are a family comprising a domain containing 10 or 12 transmembrane helices (pfam 00654).

 The first member of the CLC family that has been isolated is the CLC-0 channel. This channel was obtained by JENTSCH et al. (Nature, 348, 510-514, 1990) and comes from the electric organ of the torpedo fish. Subsequently, other CLC channels have been demonstrated in mammals, mainly in humans, rats and mice, and other animals such as Xenopus (Xenopus laevis.), Teleost fish Oreochromis mossambicus, the nematode Caenorhabditis elegans, etc. In the yeast Saccharomyces cerevisiae, only one gene (ScCLC) was detected. The systematic sequencing data indicates the presence of CLC gene homologs in many prokaryotic genomes (JENTSCH et al., Eur J. Physiol., 437, 733-795, 1999 and MADUKE et al., J. Gen. Physiol., 114, 713-722, 1999).

 In plants, several anionic channels of the CLC family have been identified to date, two channels have been identified in tobacco (LURIN et al., Plant Cell, 8, 701-711, 1996; LURIN, Thesis of the University Paris VI, Pierre and Marie Curie, France, 1998), one in the potato (CZEMPINSKI et al., J. Exp.Bot 50,955-966, 1999), seven in the model plant Arabidopsis thaliana whose entire genome was sequenced: AtCLC-α, -b, -c and -d (HECHENBERGER et al., J. Biol Chem., 271, 3632-33638, 1996); AtCLC-g (GEELEN et al., Plant J., 21, 259-269, 2000); AtCLC-e (AF 366367, GenBank, May 15, 2001) and AtCLC-f (AF 3666368, GenBank, May 15, 2001) were identified by the inventors).

 The anionic channels in plants are mainly located on the plasma membrane and the tonoplast (BARBIER-BRYGOO et al., Biochim Biophys Acta., 1465, 1999-218, 2000, HEDRICH and BECKER, Plant Mol Mol. 26, 1637-1650, 1994; TYERMAN, Annu Rev. Plant Physiol.

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 Plant Mol. Biol., 43, 351-373, 1992) and also on the membranes of mitochondria and plastids (BORECKY et al., J. Biol Chem, 272, 19282-19289, 1997, NEUHAUS and WAGNER, Biochim Biophys Acta. 1465, 307-323, 2000).

 The intervention of anionic channels in different physiological functions of the plant has been proposed: - in growth and development via hormonal signaling pathways, - in responses to biotic and abiotic stresses, in responses to microorganisms pathogens, - in mineral nutrition.

réponses aux stress (JABS et al., Proc. Natl. Acad. Sci. USA, 94, 4800-4805, 1997 ; CAZALE et al., Plant Physiol., 116, 659-669,1998). Il apparaît clairement que les canaux anioniques ont un rôle important dans la physiologie de la plante, mais que dans de nombreux cas, les données accumulées ne permettent pas encore d'associer un rôle physiologique définitif aux activités de type canal caractérisées ou d'associer un canal particulier aux différents processus impliquant des transports d'anions. However, the involvement of anionic channels has been clearly demonstrated only in a few responses such as opening and closing of stomata and in the regulation of hypocotyl elongation. In the responses to biotic and abiotic stresses, the anionic channels would intervene very much before the triggering of the

stress responses (JABS et al., Proc Natl Acad Sci USA, 94, 4800-4805, 1997, CAZALE et al., Plant Physiol., 116, 659-669, 1998). It is clear that anionic channels play an important role in the physiology of the plant, but that in many cases the accumulated data do not yet allow a definitive physiological role to be attached to the characterized channel activities or to particular channel to different processes involving anion transports.

 In Arabidopsis, most of the 7 genes encoding the AtCLC-α to -g channels are located on chromosome V, except the AtCLC-b, -e and -f genes located respectively on chromosomes III, IV and I.

 The five plant channels AtCLC-a, -b, -c, -d, and -g are close to the intracellular animal anionic channels of type CLC-6 and CLC-7. The other two members of the AtCLC-e family and AtCLC-f form another subfamily with bacterial CLCs.

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 Plant CLC genes are expressed throughout the plant although differences between organs have been demonstrated. For example, AtCLC-a is preferentially expressed in leaves, AtCLC-b in roots, AtCLC-d in leaves and roots (HECHENBERGER et al., 1996, supra publication).

phénotypes de déficience respiratoire et de sensibilité au pH dans le cas d'AtCLC-d, et uniquement pour la sensibilité au pH pour AtCLC-c (GAXIOLA et-al., Proc. Natl. Acad. Sci. USA, 95,4046-4050, 1998 ; HECHENBERGER et al., 1996, publication précitée). Cependant, aucune corrélation n'a pu être établie entre la fonction complémentée chez la levure, et la fonction réelle de ces protéines dans la plante. S'appuyant sur la localisation dans le Golgi de ScCLC chez la levure, une activité dans les endomembranes des protéines AtCLC-c et AtCLC-d a été suggérée. The function in plants of AtCLC proteins is only beginning to be elucidated. AtCLC-c and AtCLC-d are able to complement the yeast gefl mutant for

phenotypes of respiratory failure and pH sensitivity in AtCLC-d, and only for pH sensitivity for AtCLC-c (GAXIOLA et al., Proc Natl Acad Sci USA, 95,4046; 4050, 1998, HECHENBERGER et al., 1996, publication cited above). However, no correlation could be established between the complement function in yeast, and the actual function of these proteins in the plant. Based on the Golgi localization of ScCLC in yeast, activity in the endomembranes of AtCLC-c and AtCLC-d proteins has been suggested.

 GEELEN et al. (2000, supra) have shown that clca-1 plants with a mutation in the gene coding for the AtCLC-a tonoplastic anion channel subaccumulate nitrate, and also exhibit hypersensitivity to chlorate. The chlorate can enter via the nitrate transporters in the cell, where it will be converted by nitrate reductase into chlorite, a toxic compound. It seems likely that in wild plants chlorate and chlorite are accumulated in the vacuole via the AtCLC-a protein. In the coca-1 mutant plants, the absence of the AtCLC-a protein causes an accumulation of chlorate and chlorite in the cytoplasm, hence the observed hypersensitivity.

 The inventors have now found that the inactivation of the AtCLC-e gene coding for the AtCLC-e chlorine channel causes an under-accumulation of nitrate in the mutant plants. Thus, in a mutant of Arabidopsis called key-1, in which the AtCLC-e gene was inactivated by the insertion of T-DNA, an under-accumulation of

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 nitrate of about 56% compared to wild plants. This mutant also has a malate rate 1.5 times higher than that of wild plants, and a greater sensitivity to salicylic acid. On the other hand, at the level of the morphology of the floral and vegetative organs, as well as at the level of the development, its phenotype does not appear different from that of the wild plants. In addition, unlike the clca-1 mutant, it does not show hypersensitivity to chlorate.

 The AtCLC-e channel is encoded by a single copy of the AtCLC-e gene located on chromosome IV. It consists of 6 exons. AtCLC-e cDNA. Referenced in the present invention by the sequence SEQ ID NO: 1 is composed of a 5 'untranslated region of 30 base pairs, followed by an open reading phase of 2127 bp encoding a 709 amino acid protein having a mass calculated molecular weight of 75.4 kDa. The sequence of this protein is represented in the attached sequence listing under the number SEQ ID NO: 4. The 3 'untranslated region is composed of 360 bp and no potential polyadenylation signal has been found in this region.

 The inventors have furthermore studied the subcellular localization of the AtCLC-e channel, and have demonstrated a chloroplast addressing of this protein.

 The present invention relates to a method for reducing the accumulation of nitrate in a plant, characterized in that a chloroplast chlorine channel, and in particular the CLC-e channel, of said plant is inhibited.

plante. Des gènes homologues d'AtCLC-e peuvent être identifiés et clonés, selon des méthodes classiques, à partir de banques d'ADNc ou d'ADN gnomique de la plante souhaitée, à l'aide d'oligonucléotides choisis à partir de la séquence d'ADNc GenBank AF 366367, qui est également représentée dans Here, the CLC-e channel is defined not only the AtCLC-e protein encoded by the AtCLC-e gene of Arabidopsis thaliana but also any chloroplast chlorine channel encoded by a homologous gene of another.

plant. Homologous genes of AtCLC-e can be identified and cloned, according to standard methods, from cDNA or genomic DNA libraries of the desired plant, using oligonucleotides selected from the sequence of GenBank AF 366367 cDNA, which is also shown in

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 the list of sequences in the appendix under the number SEQ ID NO: 1, or the coding portion of this cDNA sequence, represented in the attached sequence listing under the number SEQ ID NO: 2.

 It will also be possible to recover genes homologous to the AtCLC-e genes in the EST libraries or in the genomic libraries of the plants of interest, the corresponding oligonucleotides may be used.

 AtCLC-e-specific oligonucleotides, which can easily be identified by comparing the sequence of this gene with the sequences of the genes encoding the other Arabidopsis thaliana CLC channels, also available on the sequence databases, will preferably be selected.

d'Arabidopsis thaliana dans lequel le gène AtCLC-e est inactivé, par exemple en vérifiant que l'expression du gène cloné dans ce mutant restaure l'accumulation du nitrate. The function of the gene cloned from the chosen plant can be controlled by complementation of a mutant

of Arabidopsis thaliana in which the AtCLC-e gene is inactivated, for example by verifying that the expression of the gene cloned in this mutant restores the accumulation of nitrate.

 By inhibition of the CLC-e channel is meant any alteration of this channel which may range from a simple decrease in its activity to total inactivation.

 This inhibition can be obtained in various ways, for example: by mutation of the CLC-e gene; this mutation may be a deletion of all or part of the gene, an insertion of an exogenous sequence (which includes the replacement of the wild-type gene sequence with a mutated gene), a point mutation, or a combination of these different mutations. Depending on the extent and location of the portions of the gene involved, the total inactivation of the gene may be reduced to a level of expression of the CLC-e protein (if the mutation is localized at the level of the control sequences). expression), or to the production of an inactive or reduced activity CLC-e channel, for example due to a transmembrane region-mediated mutation, or the chloroplast targeting sequence.

 by inhibition of the transcription or translation of the CLC-e gene, leading to the decrease of the

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 amount of protein synthesized, or lack of protein synthesis.

 by inhibiting the activity of the CLC-e protein, for example by a chemical effector absorbed by the plant.

 The mutation of the CLC-e gene can be carried out by various methods known in themselves to those skilled in the art; for example, site-directed mutagenesis can be carried out using conventional techniques, such as homologous recombination techniques, to target the portion of the CLC-e gene that is to be inactivated.

Advantageously, it will also be possible to use methods comprising a step of random mutagenesis, for example by insertion of T-DNA, and a targeted screening step allowing the detection of mutants of the CLC-e gene. Other random mutagenesis and screening techniques that can be used for the implementation of the present invention are for example described by LI et al. (Plant J., 27, 235-242, 2001), or by McCALLUM et al. (Nature Biotech., 18, 455-457, 2000).

 Mutagenesis of the CLC-e gene makes it possible in particular to obtain plants expressing an inactive channel, for example because of an alteration or deletion of the chloroplast targeting sequence.

 Inhibition of transcription or translation of the CLC-e gene can be achieved by conventional techniques, for example by co-suppression or antisense suppression, using recombinant DNA constructs producing transcripts in the cells. meaning or antisense derived from the cLC of the protein CLC-e. Useful techniques are described, for example, in the review article by MATZKE et al. (Curr Opin Genet Dev, 11 (2), 221-227, 2001) and the publications referenced therein.

 The mutation of the CLC-e gene or the inhibition of its transcription or translation can be implemented, according to the invention, by using polynucleotides derived from the sequence of said gene. The subject of the present invention is therefore the use of at least one polynucleotide comprising all or part of the

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 sequence of a CLC-e gene, or its complement, to decrease the accumulation of nitrate in a plant.

 Advantageously, a polynucleotide comprising all or part of the sequence of the cDNA of a CLC-e channel, or its complement, or at least one polynucleotide representing a fragment of at least 20, preferably at least 50, will be used. and most preferably at least 100 consecutive bases of said sequence.

 It is also possible to use, for the implementation of the present invention, polynucleotides whose sequence is not completely identical to or complementary to that of the CLC-e gene of said plant or of a fragment thereof. In this case, polynucleotides having at least 80%, advantageously at least 90%, and most preferably at least 95% identity with the polynucleotide SEQ ID NO: 1, SEQ ID NO: 2 will preferably be selected. , or SEQ ID NO: 3, or with the desired fragment thereof, or their complementary.

 The size of said polynucleotide will be chosen according to the intended use. For example, for screening mutants of the CLC-e gene, polynucleotides can be used from 20 bases. For antisense suppression, longer polynucleotides, generally comprising at least 50, and preferably at least 100 bases, will be preferred in order to allow stable hybridization with CLC-e mRNA.

 It is particularly possible to use, for the implementation of the present invention, polynucleotides of sequence SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or their fragments, as well as polynucleotides having at least 70 %, preferably at least 80%, advantageously at least 90%, and most preferably at least 95% identity with the polynucleotide SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 , or with the desired fragment thereof.

 Similarly, polynucleotides of sequence SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or fragments thereof, may be used in the practice of the present invention to obtain the inhibition of the CLC-e channel

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 in plants other than Arabidopsis thaliana, in order to reduce the accumulation of nitrate in said plants.

 The present invention more particularly relates to a method for obtaining a plant subaccumulating nitrate, characterized in that it comprises: - the transformation of a plant host cell with at least one copy of a polynucleotide chosen from: a) polynucleotides of sequence SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or their fragments of at least 50, preferably at least 100 bases; or b) polynucleotides whose sequence has at least 70%, preferably at least 80%, advantageously at least 90%, and most preferably at least 95% identity with one of the polynucleotides a) above; the regeneration of an entire plant from the host cell obtained in the previous step.

 For the implementation of the present invention, the polynucleotide used is inserted, in the sense orientation, or in the antisense orientation, according to the chosen strategy, into an expression cassette, under transcriptional control of a suitable promoter. Advantageously, said expression cassette also comprises a transcription terminator.

 The invention also encompasses the thus obtained expression cassettes.

 Among the transcription promoters that may be used, mention may be made in particular of constitutive promoters, inducible promoters, as well as tissue-specific promoters.

 A constitutive promoter allows strong expression of the transcript in all tissues of the regenerated plant and will be active in most environmental conditions, and in all stages of cellular transformations and differentiations.

 For example, constitutive promoters frequently used are:

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 the 35S promoter, or advantageously the 35S double promoter (pd35S) of CaMV, the actin promoter.

 Alternatively, it may be advantageous to use an inducible transcription promoter capable of being controlled by the environmental conditions or the stage of development, such as, for example, phenylalanine ammonia-lyase (PAL) promoters, chitinases, nopaline synthase (nos. ) or the vspB gene (US 5,670,349). By way of nonlimiting examples, mention may be made in particular of the promoters appearing on the list presented in Table 3 of US Pat. No. 5,670,349.

 Another category of promoters that can advantageously be used to carry out the process that is the subject of the invention groups tissue-specific promoters; for example, they may be promoters of genes expressing themselves specifically in the leaves, such as, for example, the promoter of the subunit gene of RUBISCO encoded by the nuclear genome.

 The transcription terminator is placed at the end 3 'of the sequence to be transcribed. It can come from the same gene as the promoter sequence or from a different gene.

 Among the transcription terminators that may be used, there may be mentioned, for example: the polyA 35S terminator of cauliflower mosaic virus (CaMV), or the polyA NOS terminator, which corresponds to the 3'-nonsense region. coding of the nopaline synthase gene of the Ti plasmid of Agrobacterium tumefaciens nopaline strain.

 Preferably, said expression cassette also comprises a selection marker for selecting the transformed plant cells. Many selection markers are known to those skilled in the art; those which confer resistance to one or more toxins, for example resistance to antibiotics such as ampicillin, spectinomycin,

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kanamycin, or resistance to herbicides such as BASTA '
Transformation of plant cells can be controlled by other techniques well known to those skilled in the art such as Southern, Northern and Western blots.

 By way of example, an expression cassette according to the invention may comprise: the strong constitutive 35S promoter of the cauliflower mosaic, a sense polynucleotide encoding the AtCLC-e anionic channel of sequence SEQ ID NO: 1 or an antisense polynucleotide corresponding to the complementary strand of the mRNA transcribed from the sequence coding for the anionic channel AtCLC-e and referenced in the present invention by the sequence SEQ ID NO: 2 - the terminator Nos corresponding to the 3 'region non-coding of the nopaline synthase gene of the Agrobacterium tumefaciens Ti plasmid nopaline strain.

 To transform the host cell, the expression cassette can be introduced into an expression vector adapted to transform the plant cells.

The expression vectors thus obtained are also part of the subject of the invention.

 As a non-limiting example of vectors that can be used to obtain expression vectors in accordance with the invention, mention may be made of the binary vectors, which are well known to those skilled in the art.

 The transformation can thus be carried out via a bacterial host such as Agrobacterium tumefaciens or Agrobacterium rhizogenes.

 Preferentially, the transformation of the plant cells is carried out by the transfer of the T region of the Agrobacterium tumefaciens Ti tumor-inducing extrachromosomal circular plasmid, using a binary system. To do this, two vectors are built. In one of these vectors, the T-DNA region was deleted except for the right and

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 left, a marker gene being inserted between them to allow selection in plant cells. The other partner of the binary system is an auxiliary Ti plasmid, a modified plasmid that no longer has T-DNA but still contains the virulence vir genes required for the transformation of the plant cell. This plasmid is maintained in Agrobacterium.

 Other transformation methods which can be used are, for example: the transformation of plant protoplasts, in particular after incubation of the latter in a solution of polyethylene glycol in the presence of divalent cations (Ca 2+); - transformation by electroporation; transforming using a particle gun that allows the projection, at a very high speed, of metal particles covered with the DNA sequences of interest, thus delivering the sequences inside the cell nucleus; the transformation by cytoplasmic or nuclear microinjection.

 The cells transformed by the constructs that are the subject of the present invention are then selected for example on the basis of their resistance to antibiotics, especially kanamycin resistance. The transgenic cells are then cultured, conventionally, on a suitable medium for the regeneration of whole plants.

 The principle of in vitro regeneration from plant cells is well known to those skilled in the art. The plants are regenerated from callus obtained after culture of anther cells, pollen grains, embryos or protoplasts. The cells are cultured on nutrient agar media adapted to each cell type under sterile conditions in petri dishes or culture tubes in a climatic chamber under favorable environmental conditions (adapted temperature and brightness).

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 In the case of protoplasts, the cell wall can reform under the effect of appropriate osmotic conditions. In the case of seeds or embryos, a favorable medium for germination or callus initiation is employed. For explants, the medium used must allow regeneration. After transplanting, vitroplants are transplanted into a greenhouse.

 The invention also encompasses host cells, especially plant cells as well as plants transformed with recombinant DNA constructs defined above.

 The subject of the present invention is also any nitrate-substituting plant capable of being obtained by inhibition of the CLC-e chloroplast channel in accordance with the invention.

 Of course, this includes the descendants, obtained by sowing or by vegetative propagation, of the plants directly obtained by the process of the invention.

 By plant under-accumulating nitrate is defined here any plant in which the rate of nitrate measured in the seedlings or in the leaves is lower than the average rate of nitrates measured in the seedlings or leaves of wild-type plants of the same species , grown under the same conditions. Advantageously, the nitrate level in the plantlets or in the leaves of a plant according to the invention is at least 20% less, preferably at least 40% less, and quite preferably at least less than 40%. 50% at the average rate of nitrates of wild-type plants of the same species.

 For the determination of the nitrate level, the skilled person may use the nitrate measuring means he deems appropriate. As an indication, the inventors measured the nitrate level in the seedlings by capillary electrophoresis (Waters), a separation technique that accurately assays the anions in small volume samples. Capillary electrophoresis measures the retention rate of anions on the chromate buffer. Retention times and peak areas are converted to

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 concentration against a standard curve. Another technique for performing a colorimetric assay of nitrate levels is based on the enzymatic reduction of nitrate to nitrite (BARTHES et al., Plant Physiol Biochem., 33, 173-183, 1995).

 Preferably, nitrate-substituting plants in accordance with the invention belong to species consumed in human or animal nutrition. These species may belong to different families, such as crucifers, solanaceae, chenopodiaceae, compounds, cucurbits, rosaceae, umbelliferae, etc.

 The invention also includes plant cells and tissues, as well as organs or parts of plants, including leaves, stems, roots, flowers, fruits, and / or seeds with a reduced nitrate content, obtained from a plant according to the invention.

 The present invention will be better understood with the aid of the additional description which follows, which refers to non-limiting examples illustrating the production and characterization of transgenic plants expressing a coding sequence for a CLC-e channel, in sense orientation. or antisense.

pSR3. D'autres plasmides ont été utilisés pour les expériences de complémentation du mutant clce-1. EXAMPLE 1 TRANSFORMATION OF PLANTS BY AGROBACTERIUM TUMEFACIENS 1 Expression Plasmids
A binary vector pSR3 was used to produce transgenic plants. Transgenic plants overexpressing AtCLC-e constructs in sense and antisense orientation under the control of the 35S promoter of cauliflower mosaic virus, were produced with the plasmid

PSR3. Other plasmids were used for complementation experiments of the clce-1 mutant.

 The plasmid pSR3 contains the ampicillin resistance gene for selection in Escherichia coli, spectinomycin for the selection of Agrobacteria and the

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 kanamycin for the selection of transformed plants. This plasmid is shown in FIG.

avec antibiotiques (SAMBROOK et al., Molecular Cloning, A laboratory manual, 2nd edition : New York, Cold Spring Harbor Laboratory Press, 1989) sont inoculées avec 200 jul d'une préculture saturée et sont mises en agitation à 30 C jusqu'à atteindre une DO à 600 nm de 0,5 à 0,6 (16 h à 18 h). Les étapes suivantes sont réalisées à 4 C en conditions semistériles. Les bactéries sont refroidies pendant 15 à 30 min sur la glace, puis récoltées grâce à une centrifugation de 15 min à 4000 rpm (Sorval, rotor JA10) à 4 C. Le culot est repris par 250 ml d'eau 1 mM Hepes. Le culot obtenu après une centrifugation de 10 min à 5000 rpm (rotor JA-10) à 4 C, est repris dans 25 ml d'eau Hepes 1 mM. Le mélange est centrifugé 10 min à 7000 rpm (JA-20) puis repris dans 25 ml d'eau Hepes 1 mM, puis à nouveau centrifugé dans les mêmes conditions et repris par 25 ml de glycérol 10%, puis par 500 jul de glycérol 10%. La suspension de bactéries est ensuite séparée en fractions aliquotes de 40 ul et conservées à-80 C. b. Electroporation des agrobactéries

40 u. l de bactéries compétentes, préparées comme décrit ci-dessus, sont mis à décongeler sur la glace. Après ajout de 60 ptl de glycérol 10% et de quelques dizaines de ng de plasmide à transférer, elles sont transvasées dans une cuve à électroporation refroidie et électroporées dans les conditions suivantes : 2500 V, 40 F, 192 Q. Rapidement 900 pal de milieu SOC (SAMBROOK et al., 1989, publication précitée) sont ajoutés aux bactéries électroporées, qui sont incubées lh30 à 30 C avec agitation, puis étalées sur milieu YEP contenant les antibiotiques de sélection. 2. Transformation by electroporation of Agrobacteria a. Preparation of electro-competent agrobacteria
Two Erlenmeyer flasks containing 300 ml of LB

with antibiotics (SAMBROOK et al., Molecular Cloning, A laboratory manual, 2nd edition: New York, Cold Spring Harbor Laboratory Press, 1989) are inoculated with 200 μl of a saturated preculture and are stirred at 30 ° C. reach an OD at 600 nm from 0.5 to 0.6 (16 h to 18 h). The following steps are carried out at 4 C under semi-sterile conditions. The bacteria are cooled for 15 to 30 min on ice, then harvested by centrifugation for 15 minutes at 4000 rpm (Sorval, rotor JA10) at 4 C. The pellet is taken up in 250 ml of water 1 mM Hepes. The pellet obtained after centrifugation for 10 min at 5000 rpm (rotor JA-10) at 4 ° C. is taken up in 25 ml of 1 mM Hepes water. The mixture is centrifuged for 10 min at 7000 rpm (JA-20) and then taken up in 25 ml of 1 mM Hepes water, then again centrifuged under the same conditions and taken up in 25 ml of 10% glycerol, then with 500 μl of glycerol. 10%. The bacterial suspension is then separated into 40 μl aliquots and stored at -80 ° C. Electroporation of Agrobacteria

40 u. 1 of competent bacteria, prepared as described above, are defrosted on ice. After addition of 60 μl of 10% glycerol and a few tens of ng of plasmid to be transferred, they are transferred to a cooled electroporation cell and electroporated under the following conditions: 2500 V, 40 F, 192 Q. Rapidly 900 μl of medium SOC (SAMBROOK et al., 1989, publication cited above) are added to the electroporated bacteria, which are incubated for 30 minutes at 30 ° C. with stirring, then spread on YEP medium containing the selection antibiotics.

3. Transformation of Arabidopsis plants
200 ml of YEP medium containing antibiotics are inoculated with 2 ml of a pre-culture made from

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 an isolated colony of agrobacteria containing the plasmid of interest, then stirred for 48 h at 300C until the suspension reaches an OD of 0.8 to 1 at 260 nm. The bacteria are harvested after centrifugation for 10 min at 5000 rpm (JA14) at room temperature. The pellet is gently taken up in 300 ml of 1% sucrose, then transferred to a magenta pot, in which 150 g of Si. Lwet L-77 (Lehle seeds), a detergent which will make it possible to prepare the plant tissues for transformation. The Arabidopsis plants of ecotype Columbia about one month, at the stage flower buds (about 30 plants per transformation), are incubated 2 min in the previous solution without evacuating. The transformed plants are then placed under a bell, lying down for the night. The next day, the plants are again placed upright, then the plants are put in the greenhouse to produce seeds from which will be selected the primary transformers.

EXAMPLE 2 BOMBARDMENT TRANSFORMATION 1 Fixation of the DNA to the gold beads
For each plasmid construct containing the AtCLC-e sense or antisense sequence, three bombardments are made. The transfer of DNA into the leaf cells is done with gold particles. The gold beads (Biorad, 1.6 μm) prepared as specified by the supplier are vortexed for 10 minutes. Fixation of the DNA on the beads is carried out as follows: to 25 l of gold beads (60 mg / ml) are added, while vortexing, 2.5 l of plasmids of interest (1 g / l) prepared using the Qiagen Kit (QIAprep Spin Miniprep), 25 μL of 2.5M CaCl 2 and 10 μM. The mixture is then vortexed for 3 minutes and then the beads are pelleted by incubation for 1 minute and centrifugation for 2 seconds at maximum speed in a benchtop centrifuge. The pellet of the beads is washed with 70 μl of 70% ethanol and then with 70 μl of absolute ethanol, then resumed by tapping with the finger on the tube and stirring with a vortex at a low speed in 60%. ethanol

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absolu. Pour chaque bombardement, 20 l de la solution de billes, associées à l'ADN sont déposés sur des macrocarriers (Bio-Rad) et mis à sécher avant bombardement.

absolute. For each bombardment, 20 l of the solution of beads, associated with the DNA are deposited on macrocarriers (Bio-Rad) and dried before bombardment.

2 jours), sur une surface d'environ 2 cm2. Les pétioles sont enfoncés dans la gélose. 2. Bombing
The bombardments are carried out with a PSD-1000 / He (Bio-Rad) device using the following parameters: vacuum 28 inches Hg, bombardment distance 6 cm, helium pressure 1350 psi. The leaves used are from Arabidopsis seedlings (Columbia ecotype) about one month old and grown in vitro on solid ABIS medium in Petri dishes (8 cm in diameter) positioned horizontally. Just before the bombardment, the leaves are removed by incision at the petiole and are transferred, adaxial faces upward, to the center of a new box of solid ABIS (medium left dried two

2 days), on an area of about 2 cm2. The petioles are embedded in the agar.

Composition of the ABIS medium

<Tb>
<tb> Composition
<tb> Concentration <SEP> final
<tb> Macroelements
<tb> K2HPO43H2O <SEP> + <SEP> KH2PO4 <SEP> 2.5 <SEP> mM <SEP>;<SEP> pH <SEP> 6
<tb> KNO3 <SEP> 5mM
<tb> MgS047H20 <SEP> 2 <SEP> mM
<tb> Ca <SEP> (N03) <SEP> 2H20 <SEP> 1 <SEP> mM
<tb> FeEDTA <SEP> 50 <SEP> ism
<tb> Microelements <SEP> (Murashige <SEP> and <SEP> Skoog, <SEP> 1962)
<tb> H3BO3 <SEP> 100 <SEP> pM
<tb> KI <SEP> 5pM
<tb> MnS044H20 <SEP> 100 <SEP> pM
<tb> ZnS04 <SEP> 7H2O <SEP> 30 <SEP>! <SEP> -lM
<tb> Na2MoO4 <SEP> 5H20 <SEP> 1 <SEP> pM
<tb> CuSO4 <SEP> 5H2O <SEP> 0, <SEP> 1 <SEP> PM
<tb> CoCI26H2O <SEP> 0.1 <SEP> (JM
<tb> MES <SEP> 1 <SEP> mM
<tb> Sucrose <SEP> 10 <SEP> g / L
<tb> Bacto <SEP> agar7 <SEP> g / L
<tb> PH <SEP> adjusted <SEP> to <SEP> 6.1 <SEP> with <SEP> of <SEP> KOH <SEP> 0.5M <SEP> before <SEP> autoclaving
<Tb>
EXAMPLE 3: SELECTION OF TRANSGENIC PLANTS
The hemizygous primary transformants were selected from kanamycin (100 mg / l) transformed plant (TO) seeds for constructs made in plasmid pSR3. Plasmid pFP101 contains GFP under the control of the seed-specific promoter, primary transformants

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 corresponding to the fluorescent seeds were selected under the binocular epifluorescence microscope. For each construction, Table 1 summarizes the different selection steps that were performed to isolate homozygous and single copy transgenic plants, as well as the number of plants selected for each generation.

 For each transformation, several primary transformants (Tl seeds) were selected and cultured in the greenhouse for selfing. Among the hemizygous plants selected in T1, those whose progeny (T2 seeds) were segregated on kanamycin (75% resistant to Kanamycin, 25% sensitive to kanamycin) were selected. For each Tl plant selected, 10 T2 plants were greenhouse-grown, self-fertilized to obtain T3 seeds and to select homozygous T2 plants for insertion carrying kanamycin resistant T3 seeds.

For each homozygous plant, five plants were put in the greenhouse to produce a seed stock for phenotypic analyzes. It is important to select several plants for each construction because in particular depending on the place of integration of the transgene in the genome it may be more or less well expressed.

 For the control transformation carried out with the plasmid pSR3, we selected 4 independent T3 plants homozygous for insertion (Table I).

 With respect to plants transformed with the pSR3AtCLCe-S construct to produce transgenic plants overexpressing AtCLC-e in the sense orientation and by the pSR3AtCLC-eAS construct to produce transgenic plants overexpressing an antisense form of AtCLC-e, we respectively have selected 7 and 5 independent homozygous T3 plants The balance of this selection is shown in Table I.

 Homozygous plants with only one copy of the transgene are selected by the kanamycin resistance marker gene. m: mutant allele, containing an insertion, +: wild allele. Gray boxes

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 correspond to the plants selected at each stage. Kanamycin-sensitive Kana5, Kanar: Kanamycin-resistant.

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<Tb>
<Tb>

Tableau 1.

Construction <SEP> pSR3 <SEP> pSR3AtCLC-eS <SEP> pSR3AtCLC-eAS
<tb> (control)
<tb> Selection <SEP> among <SEP> iesgrainesït <SEP> of
<tb> resistant <SEP> primary <SEP> transformants <SEP> to <SEP><SEP> 37/10000 <SEP> 51/6000 <SEP> 19/6000
<tb> kanamycin
<tb> Rate <SEP> Detransformation <SEP> 0.37% <SEP> 0.85% <SEP> 0.32%
<tb> Selection <SEP> of <SEP> plants <SEP> T1 <SEP> of which <SEP> la
<tb> 5/37 <SEP> 9/51 <SEP> 10/19
<tb> progeny <SEP> (T2) <SEP> shows <SEP> a <SEP> segregation <SEP> (54, <SEP> 71, <SEP> 72, <SEP> 75, <SEP> 81) <SEP > (9, <SEP> 19, <SEP> 23, <SEP> 31, <SEP> 34, <SEP> 39, <SEP> 41, <SEP> 46.47) <SEP> (2,3, <SEP> 7.9, <SEP> 10, <SEP> 15, <SEP> 17, <SEP> 18, <SEP> 19,
<tb> on <SEP> kanamycin: <SEP> 3/4 <SEP> Karf, <SEP> 1/4 <SEP> Kans <SEP> 20)
<tb> Transfer <SEP> to <SEP><SEP> greenhouse <SEP> from <SEP> 10 <SEP> plants <SEP> TZ <SEP> resistant <SEP> to <SEP><SEP> kanamycin <SEP > by <SEP> plane <SEP> T1 <SEP> selected
<tb> Selection <SEP> ection <SEP> of <SEP> plantsT2 <SEP> of which <SEP><SEP> 5 <SEP> plants <SEP> retained <SEP> 8 <SEP> plants <SEP> retained <SEP > 5 <SEP> plants <SEP> retained
<tb> progeny <SEP> (T3) <SEP> shows <SEP> 100% <SEP> of independent <SEP> independent <SEP> independent <SEP>
<tb> resistance <SEP> to <SEP><SEP> kanamycin, <SEP> therefore <SEP> 54-10, <SEP> 71-7, <SEP> 19-8, <SEP> 31-4,34 -5, <SEP> 2-2, <SEP> 3-7
<tb> homozygous <SEP> for <SEP> insertion <SEP> 72-3,75-5 <SEP> 39-1, <SEP> 41-9, <SEP> 7-6,9-6
<tb> 81-2 <SEP> 46-6.46-10, <SEP> 47-4 <SEP> 19-4
<tb> Transfer <SEP> to <SEP><SEP> greenhouse <SEP> from <SEP> 5 <SEP> plants <SEP> T3 <SEP> with <SEP> plant <SEP> T2 <SEP> selected
<tb> Production <SEP> of <SEP> seeds <SEP> 4 <SEP> plants <SEP> independent <SEP> 7 <SEP> plants <SEP> independent <SEP> 5 <SEP> plants
<tb>SEP> molecular <SEP> and <SEP> phenotypic analysis <SEP> 54-10-5, <SEP> 71-7-2, <SEP> 19-8-4, <SEP> 31-4- 2, <SEP> 34-5-2, <SEP> independent
<tb> on <SEP> the <SEP> progeny <SEP> (T4) <SEP> of a <SEP> plant <SEP> 75-5-2 <SEP> 81-2-5 <SEP> 39-1 -5, <SEP> 41-9-2.46-10-4, <SEP> 2-2-5, <SEP> 3-7-1, <SEP> 7-6-3,
<tb> T3 <SEP> on <SEP><SEP> 5 <SEP> set <SEP> to <SEP> on <SEP> within <SEP> 47-4-4 <SEP> 9-6-3, <SEP> 19-4-3
<tb> Homozygotes <SEP> for <SEP> insertion
<Tb>
AtCLC-e

Table 1.

Genotype n1 "1+ 1 Self-fertilization, S '! P!'.

25% + / + I Self-fertilization 50% m / + 25% m / m 25% + / + or. OQm Self-fertilization qc'm'ynn

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EXAMPLE 4: COMPLEMENTATION OF THE MUTATION clce
In order to demonstrate the binding between the mutant phenotype and the insertion of the T-DNA into the AtCLC-e gene, the complementation of mutant homozygous clce-1 plants that have undergone two backcrosses using a construct expressing AtCLC-e cDNA under the control of the strong 35S promoter of cauliflower mosaic virus was performed. Recombinant ASE agrobacteria were obtained by electroporation and were used for transformation of about 30 clc-1 mutant plants at the flower bud stage according to the transformation technique based on the use of Silwet as a detergent. Control plants were produced following the transformation of mutant key-2 homozygous seedlings with agrobacteria containing the empty plasmid.

EXAMPLE 5: PHENOTYPIC ANALYSIS OF TRANSFORMANTS 1. Assay of internal anions of seedlings by capillary electrophoresis
100 mg of 14-day whole seedlings grown in vitro on ABIS medium or an equivalent mass of roots and aerial parts separately are harvested in an eppendorf tube (1.5 ml), ground with a plunger and diluted 1/10 with the water. The samples are then frozen, thawed and vortexed. This step is carried out three times, then the samples are centrifuged, the supernatant is filtered and then diluted 10 times. Assay of different inorganic and organic anions is performed by capillary electrophoresis (Waters), a separation technique that allows accurate anion determination in low volume samples. The solution to be dosed is injected at the end of a large (60 cm) but small diameter (60 μm) capillary using the force of gravity to "siphon". The parameters of height and duration are constant and make it possible to inject a volume of solution always identical. A voltage of 20000 Volts is then imposed through the capillary, causing the

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d'un flux en sens opposé nommé' d'un flux en sens opposé nommé flux électroosmotique (FEO) dû aux charges négatives tapissant la paroi interne du capillaire de verre. L'OFM, fortement positif, permet de neutraliser ces charges et d'inverser ainsi le sens du flux électroosmotique, ce qui accélère de façon considérable la vitesse de migration des anions sans modifier la qualité de la séparation. La mesure proprement dite s'effectue à l'autre extrémité du capillaire. On enregistre la diminution d'absorption du chromate lors du passage des anions devant la fenêtre de lecture. Le temps de rétention et l'ordre de passage de chacun des ions sont bien caractérisés. Une gamme étalon permet de transformer les surfaces des pics en concentrations. L'anion bromure est utilisé comme étalon temporel. migration of the charges according to their sign in one direction or the other. The anions migrate through a special buffer composed of chromate (4.6 mM), absorbing light at 254 nm, and OFM (Osmotic Flow Invert) which is a cationic surfactant. The migration force of the ions linked to their charge is counteracted by the presence in the capillary

a flow in the opposite direction named 'a flow in opposite direction called electroosmotic flow (FEO) due to negative charges lining the inner wall of the glass capillary. The OFM, which is strongly positive, neutralizes these charges and thus reverses the direction of the electroosmotic flow, which considerably speeds up the migration speed of the anions without modifying the quality of the separation. The measurement itself is carried out at the other end of the capillary. The decrease in chromate absorption is recorded when the anions pass in front of the read window. The retention time and the order of passage of each of the ions are well characterized. A standard range makes it possible to transform peak areas into concentrations. The bromide anion is used as a time standard.

électrophorèse capillaire et à 15 ul de solution à doser sont ajoutés 100 ul d'une solution contenant la nitrate réductase (0, 15 U/ml ; Aspergillus/Bonringer) et un mélange d'EDTA 0,84 mM, de K2HP04+KH2P04 83,7 mM, pH 7,4 et de NADPH 0,28 mM. Le mélange est incubé à 37 C pendant 30 minutes, temps au cours duquel le nitrate est réduit en nitrite. La réaction est arrêtée avec 10 ul d'une solution 1,5 M ZnS04. 2. Rapid Colorimetric Assay of Internal Nitrate of Plantlets by Enzymatic Reduction
The nitrate assay technique by enzymatic reduction according to (BATHES et al., 1995, publication cited above) allows a simple and rapid colorimetric assay. The seedlings are prepared as for the dosages by

Capillary electrophoresis and 15 μl of the solution to be determined are added 100 μl of a solution containing nitrate reductase (0.15 U / ml, Aspergillus / Bonringer) and a mixture of 0.84 mM EDTA, K 2 HPO 4 + KH 2 PO 4. , 7 mM, pH 7.4 and 0.28 mM NADPH. The mixture is incubated at 37 ° C. for 30 minutes, during which time the nitrate is reduced to nitrite. The reaction is stopped with 10 μl of a 1.5M ZnSO4 solution.

The colorimetric determination of the nitrite is carried out by the addition of 200 μl of DC solution (0.5% sulfanylamide, 0.75 N HCl, 0.01% N-naphthyl ethylene diamine dichlorohydrate). After

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 5 minutes, the pink color proportional to the nitrite concentration can be read at an OD of 550 nm.

A reference curve referred to for the determination of each sample is made by measuring the OD for known nitrate concentrations. These reactions are carried out in Elisa plates for easier reading by spectrophotometer. The results are usually based on the weight of fresh material.

Claims (12)

1) Process for reducing the accumulation of nitrate in a plant, characterized in that the CLC-e chloroplast chlorine channel of said plant is inhibited.  2) Process according to claim 1, characterized in that the inhibition of the CLC-e channel is obtained by a method chosen from the mutation of the CLC-e gene, the inhibition of the transcription or translation of said gene, or the inhibition of the activity of the CLC-e protein.  3) Process according to claim 2, characterized in that the inhibition of the CLC-e channel is obtained by the expression in said plant of at least one polynucleotide chosen from: a) polynucleotides comprising all or part of the sequence of a CLC-e gene, or its complement; b) polynucleotides having at least 70% identity with a polynucleotide defined in a) above.  4) Process according to claim 3 characterized in that the polynucleotide comprising all or part of the sequence of a CLC-e gene is chosen from polynucleotides of sequence SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO : 3, and their fragments of at least 20 consecutive bases.  5) Process for obtaining a plant subaccumulating nitrate, characterized in that it comprises: the transformation of a plant host cell with at least one copy of a polynucleotide as defined in any one of claims 3 or 5; 4; the regeneration of an entire plant from the host cell obtained in the previous step.  6) Use of a polynucleotide as defined in any one of claims 3 or 4 to reduce the accumulation of nitrate in a plant.  7) An expression cassette comprising a polynucleotide as defined in any one of claims 3 or 4 under transcriptional control of a suitable promoter. <Desc / Clms Page number 26>  8. Nucleic acid vector comprising an expression cassette according to claim 7.  9) Plant cell transformed by an expression cassette according to claim 7.  Nitrate accumulating plant obtainable by a process according to any one of claims 1 to 5.  11) A plant according to claim 10, characterized in that the nitrate level in the seedlings or in the leaves of said plant is at least 20% lower than the average rate of nitrates of wild-type plants of the same species.  12) Plant according to any one of claims 10 or 11, characterized in that it belongs to a family chosen from crucifers, solanaceae, chenopodiaceae, compounds, cucurbitaceae, rosaceae, and umbelliferae.