EP1971689A2 - Gene promoters that can be used in plants - Google Patents

Abstract

The present invention relates to nucleic acid sequences which have a transcriptional promoter activity preferentially in the phloem of plants under conditions of stress, or in the roots, to derived sequences, to constructs containing such sequences, and also to cells transformed with said constructs and to transgenic plants. The present invention makes it possible to place any transgene under the transcriptional control of a promoter, the activity of which is tissue-specific, organ-specific and/or inducible by environmental factors, such as biotic or abiotic stresses.

Description

 Gene promoters that can be used in plants

In the agricultural field it may be interesting to be able to increase or decrease the expression of genes of interest in certain tissues of a plant and / or in response to environmental factors such as biotic or abiotic stresses that influence on the productivity of agricultural crops.

The present invention relates to novel transcriptional regulatory sequences identified in celery, whose activity is tissue-specific, organ-specific and / or inducible by environmental factors, such as biotic or abiotic stresses, and their use for transforming plants and put a gene of interest under their control.

For example, salinity is one of the most severe environmental factors limiting the productivity of agricultural crops. Although soil salinity is largely prior to agriculture, the problem has been exacerbated by agricultural practices such as irrigation. At present, about 20% of the world's cultivated areas and almost half of the irrigated areas are affected by salinity.

In addition to a huge financial cost, salinity has other serious impacts on infrastructure, water supplies, and the social structure and stability of human communities.

The responses to salinization have been twofold: (i) to introduce environmental management to control the increase of salt in the soil, through the management of irrigation and drainage, (ii) to use genetic engineering on plants to increase their tolerance to salt.

The present invention relates in particular to the second axis of researching and understanding mechanisms that allow some plants to better tolerate salt and stress in general, to develop strategies that make plants more tolerant to salt or other stress .

Plants attached to their substrate are subjected throughout their life to many variations in the environmental conditions they face by adapting their growth and development. Abiotic stress is a significant change in chemical or physical environmental factors, while biotic stress is induced by an interaction between the plant and a living organism. Abiotic factors, which particularly affect crop yields, are water stress (drought or excess water), extreme temperature changes, deficits in soil minerals and high concentration of salts or heavy metals in the soil. ground.

Saline stress caused by excessive concentration of NaCi in the plant environment therefore falls into the category of abiotic stress. Although Na ⁺ is necessary for some plants, particularly for halophytes, a high concentration of NaCl is a limiting or even toxic factor for plant growth. This phenomenon is widespread on large arable surfaces around the world.

In response to environmental stresses, plants have developed a panel of physiological and biochemical strategies to adapt or at least tolerate stress conditions. These strategies are linked to changes in gene expression, as evidenced by changes in the amounts of newly synthesized mRNA and protein. The identified genes encode proteins associated with many functions such as ion compartmentalization, redox potential equilibrium, protein degradation or protection, osmolyte synthesis. Numerous studies have been conducted in relation to molecules called "osmolytes" because their synthesis is increased in many abiotic stresses (Hasegawa et al 2000a and b). These molecules can accumulate at high levels in the salt response (or in osmotic stress), thus restoring the water balance in the cell.

It has been proposed that metabolic engineering may play a major role in increasing plant tolerance to stress. Plants transformed to express enzymes leading to the synthesis of certain osmolytes have been shown to be more resistant to salt stress than non-transformed plants (Tarczinsky et al., 1993, Shen et al., 1997a and b), thus confirming the importance of osmolytes. However, some of these results are controversial as osmolyte levels in transgenic plants were too low to explain osmotic effect (Karakas et al., 1997). This effect has therefore been attributed to an antioxidant role rather than a purely osmolyte role.

The synthesis of such osmolytes takes place in source tissues or is based on molecules derived from photoassimilates. Therefore, the long-range transport of photoassimilates into the phloem is certainly affected during salt stress, although few data are available on this point (Noiraud et al., 2000).

The elaborate phloem sap also carries a wide range of ions, metabolites and macromolecules such as proteins and nucleic acids, many of which are involved in signaling. The phloem can therefore be considered as the major player in the communication between tissues in vascular plants (Ruiz-Medrano et al., 2001). However, to date, there is little knowledge of the genes specifically expressed in the phloem or in the roots, especially in vegetable or market garden species such as celery, despite recent work on different models of plants (Vilaine et al. al., 2003). Knowledge about the promoters of these genes, especially their phloem-specific, root-specific and stress-inducible properties, is even fewer or nonexistent.

In the absence of such knowledge, it is therefore not possible to put any coding sequence under the control of a promoter which makes it possible to express this coding sequence only under stress conditions, preferentially in the phloem or in the roots.

The present inventors have now determined the sequence of three promoters whose activity is preferentially in the vascular tissues and more particularly the phloem, or the roots, this activity being, if appropriate, a function of the stress conditions of the cell. The inventors have also carried out constructions using these promoters, making it possible to express sequences of interest only under stress conditions, preferably in the phloem, or preferentially in the roots. Accordingly, the present application relates, according to a first aspect of the invention, to a nucleic acid sequence having a transcriptional promoter activity, such that said sequence comprises SEQ ID NO: 1, 2 or 3, or a fragment or fragments (or parts thereof) of at least one of these sequences. A fragment or part of SEQ ID No. 1, 2 or 3 is defined as a sequence comprising at least 30 consecutive nucleotides of SEQ ID No. 1, 2 or 3. Part of SEQ ID No. 1, 2 or 3 may contain only 30 nucleotides, but it may advantageously contain at least 50 consecutive nucleotides of SEQ ID No. 1, 2 or 3, for example exactly 50, or more than 50, or well over 75, 80 or 90. A part may also contain more than 100 consecutive nucleotides of SEQ ID No. 1, 2 or 3, in particular 120, 150 or even 180 or 200. According to the present invention, parts of SEQ ID No. 1, 2 or 3 which are preferred are fragments corresponding to almost all of SEQ ID No. 1, 2 or 3, with only the deletion of 1 to 10, 20, 30 or 50 nucleotides at the 3 'and / or 5' ends or within SEQ ID sequences No. 1, 2 or 3. It is also envisaged that a sequence according to the invention comprises at least two fragments of SEQ ID No. 1, 2 or 3, at least one of which is s fragments with a minimum length of 30 nucleotides. The different fragments may be fragments derived from distinct sequences, for example a fragment of SEQ ID No. 1 and a fragment of SEQ ID No. 2, or fragments of the same sequence. In the latter case, the fragments are preferably non-consecutive, for example separated by 2, 10, 20 or 50 nucleotides.

According to a preferred embodiment of the invention, a sequence according to the invention comprises or consists of the entirety of one of the sequences SEQ ID No. 1, 2 or 3.

By sequence having a transcriptional promoter activity is meant a sequence exhibiting a promoter activity, that is to say, of a nature to promote the transcription of a sequence placed downstream of said promoter sequence, optionally in the presence of co-factors. adequate. Such transcriptional promoter activity can be assayed by cloning a sequence capable of exhibiting such activity upstream of any sequence to be transcribed, in the presence of RNA polymerase and ribonucleotides. If RNA molecules are obtained, whereas no RNA molecule is obtained in the absence of cloning of the test sequence, then it can be deduced that said test sequence has a transcriptional promoter activity. An adequate test is described in the experimental part.

The transcription initiation site may be located either within a sequence according to the invention, or at the 3 'end of a sequence according to the invention or downstream on the 3' side of a sequence. according to the invention. In the first case, part of the sequence according to the invention is transcribed. In addition to all or part of one of the sequences SEQ ID No. 1, 2 or 3, a sequence according to the invention may also contain additional sequences, insofar as they do not suppress the transcriptional promoter property. Such additional sequences may in particular be "enhancers", or other sequences such as binding sites for different proteins. Said transcriptional promoter activity may be manifested in any type of cell, in vivo, and / or also in vitro in solution, in the presence of RNA polymerase and all the elements necessary for transcription, especially in the presence of ribonucleotides. Advantageously, a sequence according to the present invention has a ubiquitous promoter activity, that is to say both in prokaryotic and eukaryotic cells, in plant or animal cells, or in the presence of an RNA polymerase of one of these cells. A sequence according to the present invention is preferably active as a promoter in plant cells, or in the presence of an RNA polymerase from a plant cell.

A preferred property of a sequence according to the invention is its ability to promote transcription in a plant cell in response to a stimulus. Said stimulus is preferably a stimulus related to stress. Preferably, the transcriptional promoter activity possessed by a sequence according to the invention is sensitive to a biotic or abiotic stress; this activity is induced or increased under conditions of biotic or abiotic stress.

Biotic stress is the stress of a plant that is induced by an interaction between the plant and a living organism, for example during an attack of aphids.

Abiotic stress refers to significant changes in chemical or physical factors in the environment. Abiotic factors, which particularly affect crop yields, are water stress (drought or excess water), extreme temperature changes, deficits in soil minerals and high concentration of salts or heavy metals in the soil. ground.

Stress can be felt at the cellular level, or at the level of an organ of the plant, or at the level of the body as a whole.

The transcriptional activity of a sequence according to the invention can also be induced when certain components or conditions related to stress are reproduced. For example, the transcriptional activity of a sequence according to the invention can be induced following the presence of certain factors which are related to stress, such as stress proteins.

According to a preferred embodiment, a sequence according to the invention has a transcriptional promoter activity preferentially in the vascular tissues. Particularly concerned vascular tissues are phloem and / or xylem, but preferably phloem.

According to another preferred embodiment, a sequence according to the invention has a specific transcriptional promoter activity of one or more organs, that is to say a promoter whose activity is more important in certain organs of the plant. for example, stems, leaves or roots, even limited to these organs. In a preferred embodiment, a sequence according to the invention has a root-specific transcriptional promoter activity.

The transcriptional promoter activity presented by a sequence according to the invention is preferably manifested in plant cells and more particularly in cells of market garden or vegetable plants, that is to say plants used for the individual or intensive production. vegetable herbs and certain fruits (such as melon or watermelon), including Apiaceae, Asteraceae, Brassicaceae (or Cruciferae), Chenopodiaceae, Cucurbitaceae, Poaceae, Rosaceae, Solanaceae, Valerianaceae or Leguminosae. More particularly, transcriptional promoter activity occurs in cells of celery (Apium graveolens L), Arabidopsis thaliana or tomato (Solanum lycopersicum L). The sequences according to the invention comprise SEQ ID No. 1, 2 or 3 or one or more fragments (or parts thereof) of at least one of these sequences. Preferably, the sequences according to the invention consist of one of the following sequences: SEQ ID No. 1, 2, 3, 4, 5 or 6, or they comprise one of these sequences. SEQ ID NO: 4 is a sequence comprising SEQ ID NO: 1, SEQ ID NO: 5 is a sequence comprising SEQ ID NO: 2 and SEQ ID NO: 6 is a sequence comprising SEQ ID NO: 3. According to a second aspect of the present invention, a sequence according to the invention may also be a sequence which has at most 5 point mutations with respect to a sequence as defined above according to the first aspect, that is to say relative to to a sequence comprising SEQ ID No. 1, 2 or 3 or one or more fragments (or parts thereof) of at least one of SEQ ID No. 1, 2 or 3, wherein said fragment comprises at least 30 nucleotides consecutive sequence of SEQ ID No. 1, 2 or 3. A sequence according to this second aspect of the invention may for example have a single mutation with respect to a sequence according to the first aspect, but preferably at least two point mutations or 3 , 4 or 5 point mutations.

By point mutation is meant a single modification of a single nucleic acid, said modification may be the deletion of a nucleic acid with respect to the sequence without mutation, or the addition of a nucleic acid with respect to the sequence without mutation or the modification of a nucleic acid with respect to the sequence without mutation. By modification of a nucleic acid, it is meant both the substitution of a nucleic acid by another natural nucleic acid (for example substitution of A in G) and the chemical modification of a natural nucleic acid (for example the replacement adenine to 2-methyl adenosine or 4-acetylcytidine). The various modified nucleic acids that can be incorporated into a sequence according to the invention are well known to those skilled in the art.

According to one particular embodiment, a mutation may be introduced in order to suppress the transcriptional promoter activity of a sequence according to the first aspect of the invention, or to modify its characteristics.

Preferably, a sequence according to this second aspect of the invention has at most 5 point mutations with respect to a sequence comprising a fragment of SEQ ID No. 1, 2 or 3 of at least 40 consecutive nucleotides, preferably from less than 50 consecutive nucleotides. A sequence according to this second aspect of the invention may also possess transcriptional promoter activity, but not necessarily. The mutations introduced with respect to the sequence according to the first aspect of the invention may make it possible to modify the promoter activity of such a sequence, for example by modifying its initiation rate per second, or by modifying its cell specificity or transcription initiation conditions. According to a third aspect, the present invention also relates to a nucleic acid sequence which has a transcriptional promoter activity and which has at least 70% identity with a sequence selected from SEQ ID No. 1, SEQ ID No. 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. Preferably, the percentage identity is greater than 70%, that is to say at least 75 or 80%, but preferably at least 85 or 90% identity, or even at least 95% identity. Sequences also envisaged have 97%, 98%, 99%, or even 99.5% identity with one of the sequences SEQ ID Nos. 1 to 6. The percentage identity between two sequences S1 and S2 of nucleic acids is calculated by aligning the two sequences in such a way as to maximize the common sequences, possibly by inserting holes ("gap") and then dividing the total number of nucleic acids common by the number of nucleic acids of the longest sequence.

A high percentage of identity also results in the hybridization of single-stranded S1 and S2 sequences under conditions of high stringency. Conditions ensuring high stringency are well known to those skilled in the art. Indeed, stringency is defined by the experimental conditions of temperature, pH and ionic strength allowing molecular hybridization. Two parameters condition in particular the stringency: the temperature and the salt concentration. High stringency conditions correspond for example to a high temperature (above the

Tm) and / or low ionic strength, to favor specific pairings.

Conditions of high stringency can be illustrated, for example, by a medium having the properties described in the experimental part.

By "sequence according to the invention" is meant both a sequence according to the first, the second or the third aspect of the invention.

A sequence according to the invention may be in particular double-stranded DNA, but also single-stranded DNA or partially double-stranded DNA. Moreover, a sequence according to the invention can have very varied topologies, it can also be circular DNA, possibly supercoiled.

The present invention also relates to a sequence complementary to one of the previously described sequences.

A sequence according to the invention can be used in various technical fields and particularly in agriculture, especially in the transgenic plant sector.

The invention also relates to nucleic acid probes such that said probes are capable of hybridizing under conditions of high stringency with a sequence chosen from the sequences SEQ ID No. 1, 2 and 3. A probe as mentioned above comprises at least 25 nucleotides, preferably at least 30. Generally, however, the nucleic acid probes comprise less than 150 and even less than 100 or even less than 50 nucleic acids.

Such probes may make it possible to identify sequences homologous to one of the sequences

SEQ ID No. 1, 2 or 3 in different organisms, or to detect the presence of one of these sequences, in vitro or in vivo. Such probes may be single or double stranded DNA or RNA probes. They can be used, for example, as primers in PCR experiments or in a Southern blot.

The present invention also relates to a DNA construct, or DNA construct, which consists of or comprises a sequence having a promoter activity as described above and which also comprises a nucleic acid sequence to be transcribed. The construct according to the invention is made in such a way that the sequence of interest to be transcribed is situated downstream of the promoter sequence, that is to say at the 3 'end of the promoter sequence, and the transcription of the sequence of interest is directed by the promoter sequence. The sequence according to the invention and the The sequence to be transcribed may be contiguous or may be separated by intervening nucleic acids. In the latter case, the spacer sequence preferably comprises less than 200, or even less than 50 or less than 20 nucleotides.

A construct according to the invention may also comprise sequences allowing the termination of the transcription, or even sequences allowing the termination of the translation (stop codon).

Transcription of a nucleic acid sequence is said to be directed by a promoter when said promoter allows transcription of the sequence and controls that transcription. The sequence of interest is therefore transcribed under conditions and at a rate that is a function of the promoter.

It is therefore possible, by means of the teaching of the invention, to place a sequence of interest under the control of a promoter sequence of the invention so that said sequence of interest is transcribed only under conditions stress, for example only under conditions of salt stress in plant cells, or specifically at a plant organ, such as roots, or a tissue, such as phloem or and xylem . Because of the specific properties of the promoter sequences of the invention, it is therefore possible to obtain the transcription of a sequence of interest preferentially in vascular cells, and preferably under stress conditions or even more preferentially at the root level.

According to a preferred embodiment, a sequence of interest is placed under the control of SEQ ID No. 1 or SEQ ID No. 3 or a sequence according to the invention derived from one of these two sequences and having a transcriptional promoter activity, so that the transcription of said sequence of interest is induced in response to stress, especially salt stress, and more particularly at the level of the phloem of the plant.

According to another preferred implementation, a sequence of interest is placed under the control of the

SEQ ID No. 2 or a sequence according to the invention derived from SEQ ID No. 2 and having a transcriptional promoter activity, so that said sequence of interest is transcribed more importantly at the phloem level of the plant.

According to another preferred implementation, a sequence of interest is placed under the control of the

SEQ ID No. 1 or a sequence according to the invention derived from SEQ ID No. 1 and having a transcriptional promoter activity, so that said sequence of interest is transcribed more significantly at the roots than at the level of stems and leaves.

According to a particular embodiment of the invention, the promoter sequence and the sequence of interest to be transcribed that are within said construct, are heterologous with respect to each other. By heterologous means that they come from different sources, for example different organisms. It will also be said that two sequences are heterologous when at least one of the two sequences is artificial (i.e., not present in nature). The construct comprising a sequence to be transcribed heterologous with respect to a promoter sequence according to the invention therefore comprises a chimeric gene or chimeric gene. Preferably, the sequence to be transcribed is other than the sequence to be transcribed naturally associated with the promoter sequence according to the invention. According to a most preferred implementation, the sequence of interest to be transcribed is a coding sequence, which therefore is not only to be transcribed, but also to be translated. It may be an integrally coding sequence, for example a cDNA sequence, or a partially coding sequence, for example a sequence comprising introns and exons. The protein or peptide encoded by such a sequence of interest may be for example a herbicide or antibiotic resistance protein, or a growth factor, a stress resistance factor, a toxic or lethal protein. According to its needs, those skilled in the art will know which sequence of interest to place under the control of a promoter according to the invention.

These are preferably sequences encoding peptides of interest in the plant field, including peptides or proteins having activity in plant cells, or having a nutritional or aesthetic interest. It can also be lethal proteins, to remove any plant that has been stressed. It may also be a protein or a peptide allowing the detection of cells expressing it.

By DNA construct, in the context of the present invention is meant any non-natural DNA support. Such a construct can be in particular a vector allowing the transfer of the construct in a cell. The construct is preferably a vector, for example a viral vector but preferably a plasmid or plasmid vector. An advantageous plasmid in the context of the present invention is the Ti plasmid of Agrobacteria, or a plasmid derived from the Ti plasmid, which has retained the DNA transfer properties but lacks oncogenic genes.

A plasmid according to the present invention may comprise, in addition to the promoter sequence according to the invention and a sequence of interest to be transcribed, resistance genes making it possible in particular to make positive or negative selections. The said resistance genes may be resistance genes to herbicides or to antibiotics.

It may also be advantageous for the construct or the plasmid according to the invention to comprise bacterial resistance genes, for example to facilitate earlier stages of multiplication in bacteria.

The invention also relates to a plant cell which has been transformed by a sequence according to the invention or by a construct as described above, in particular by a construct comprising a sequence to be transcribed heterologous with respect to a promoter sequence according to the invention, whatever the means used to effect the transformation. Indeed, at present, very diverse means are known to allow the transformation of cells by a nucleic acid sequence. By way of example, mention may be made of transformation by electroporation, by bombardment and by the use of agrobacteria. It is obvious that, depending on the type of cells to be transformed and the species in question, particularly depending on the plant species, some techniques are preferred over others. The person skilled in the art knows, for each cell type, which are the most appropriate techniques for carrying out a transformation.

Similarly, it is well known to those skilled in the art what are the techniques which make it possible to obtain a transient transformation of the cell, the transferred genetic material being lost thereafter, and what are the techniques which allow the integration of the sequence transferred stably, in the genome of the cell. By genome of the cell is meant both the nuclear genome and the chloroplast or mitochondrial genome. Preferably, it is the nuclear genome.

The cell may be any type of cell, prokaryotic or eukaryotic, although eukaryotic cells are preferred in the context of the present invention. A cell according to the invention is also preferably a plant cell, but it can also be a bacterial cell, an animal cell, for example a mammalian cell or any other type of cell, for example a cell of yeast. Preferably, it is a plant cell of agronomic interest.

The present invention also relates to a transgenic plant comprising in its genome a sequence according to the invention, said sequence being of exogenous nature. The term "transgenic plant" and "exogenous sequence" means that the sequence according to the invention has been deliberately transferred to the plant and that said sequence was not present naturally before in the plant.

A transgenic plant according to the invention may also comprise a construct as described, in particular a construct comprising a sequence to be transcribed heterologous with respect to a promoter sequence according to the invention.

A transgenic plant according to the invention thus comprises in its genome a sequence (or a construct) of the invention; preferably, said sequence (or construct) is inserted into the nuclear genome of any cell of the plant, but the invention also includes situations in which the sequence is inserted into the mitochondrial genome or the chloroplast genome.

It is also possible to maintain the sequence or construct according to the invention extrachromosomally.

The sequence (or the construct comprising said sequence) is preferably inserted stably, although it is also possible to insert it transiently.

The present invention also relates to transgenic plants comprising transformed cells as described above.

Preferably, all of the cells of a transgenic plant according to the invention comprise a sequence or a construct according to the invention. It is also conceivable that only certain parts of these plants comprise such transformed cells, for example in the case of chimeric plants or excision of the transgene in certain cells.

The invention also relates to parts of said transgenic plants. Particularly interesting parts include fruits, flowers, roots, stems, leaves, but also seeds, buds, seeds and reproductive material, including male reproductive material and female reproductive material, as well as the callus, said parts according to the invention having transformed cells comprising a sequence or a construct according to the invention. Preferably, they comprise a construct comprising a sequence to be transcribed heterologous with respect to a promoter sequence according to the invention. These parts are therefore also transgenic.

A transgenic plant according to the invention can be any type of plant. It may be a monocotyledonous or dicotyledonous plant. Preferably, a transgenic plant is of interest agronomic, it may include a cereal plant, a vegetable or vegetable plant, a fruit tree. Preferably, it is a plant other than celery.

Among the plants, the plants of the following families are particularly preferred: the plants of the family Cucurbitaceae, Chenopodiaceae, Cruciferaceae, Poaceae, Leguminosae, Apiaceae, Rosaceae, Valerianaceae, Solanaceae and Asteraceae.

Particularly preferred examples of transgenic plants according to the invention are tomato plant, melon plant and lettuce. Other preferred herbs are celery, onion, beetroot, broccoli, wheat, asparagus, sweet corn and rapeseed.

A transgenic plant according to the present invention may also contain in its genome other transgenes, independently of the sequence according to the invention, it may be in particular a gene for resistance to a viral infection under the control of a promoter. constitutive. In this case, the different transformations may have been performed simultaneously, during a single transformation step, or sequentially.

A transgenic plant according to the invention may have been regenerated from transformed cells. It is also possible to obtain a plant according to the invention by the offspring of another transgenic plant according to the invention.

The present invention also relates to a transgenic plant comprising in its genome a nucleic acid sequence comprising all or part of SEQ ID No. 1, such that said sequence has a transcriptional promoter activity, said part comprises at least 30 consecutive nucleotides of SEQ ID NO: 1, and said sequence is in functional association with a heterologous coding sequence and expressing said coding sequence specifically in the roots. Preferably, said plant belongs to the family Apiaceae, Asteraceae, Brassicaceae (or Cruciferae), Chenopodiaceae, Cucurbitaceae, Poaceae, Rosaceae, Solanaceae, Valerianaceae or Leguminosae.

The present invention also relates to a transgenic plant comprising in its genome a nucleic acid sequence comprising all or part of SEQ ID No. 1, 2 or 3 such that said sequence has a transcriptional promoter activity, said part comprises at least 30 consecutive nucleotides of SEQ ID NO: 1, 2 or 3, and said sequence is in operative association with a heterologous coding sequence and expressing said coding sequence specifically in the phloem. In a particular embodiment, said sequence is SEQ ID 2 or a part of SEQ ID 2 comprising at least 30 consecutive nucleotides, and said plant belongs to the Apiaceae, Asteraceae, Brassicaceae, Chenopodiaceae, Convolvulaceae family. , Cucurbitaceae, Fabaceae, Grossulariaceae, Lamiaceae, Liliaceae, Poaceae, Polygonaceae, Rosaceae, Solanaceae or Valerianaceae. In another particular embodiment, said sequence is SEQ ID 1 or 3 or a part of SEQ ID 1 or 3 comprising at least 30 consecutive nucleotides, said plant belongs to the family Brassicaceae, and the expression of said sequence coding is induced by biotic or abiotic stress. In a preferred embodiment, said biotic or abiotic stress is salt stress. The present invention also relates to a method for obtaining a transgenic plant according to the invention. Such a method comprises the following steps: obtaining a construct according to the invention as described above, introducing the construct into a cell from a plant of interest and regenerating a transgenic plant, from the transformed cells. A method according to the invention can of course include many other steps, preceding or following the steps mentioned. A method according to the invention may advantageously also comprise an additional step of crossing the transgenic plant obtained with other plants, also transgenic or not. Additional crossings can of course be made. We can also carry out sexual or asexual multiplication steps, depending on the species, and thus obtain progeny.

Preferably, the progeny obtained at the end of the process is examined to determine the progeny plants that comprise a sequence or construct according to the invention. Plants comprising a sequence or a construct according to the invention can also be isolated. Such plants, which comprise in their genome a sequence or a construct according to the invention and which come from the process as described, also form part of the invention. Preferably, the plants derived from the progeny and which comprise a sequence or a construct according to the invention are determined by a selection step. Said selection may be a selection in the field or greenhouse, or a genetic selection using genetic markers.

The present invention also relates to the use of a sequence or a construct according to the invention for the production of transgenic plants, that is to say in the field of genetic engineering.

Indeed, as explained above, one application of the present invention is the use of promoter sequences of the invention positioned upstream of a coding sequence of interest (transgene), so that the protein encoded by the sequence of interest is only expressed in specific stress conditions, and preferentially in some vascular organs of the transgenic plant. By this means, the transgene is little or not expressed under normal conditions and its expression is induced only under stress conditions. This characteristic of the invention is of great interest since it becomes possible to express certain transgenes, especially resistance genes, only when it is necessary, that is to say under stress conditions. Indeed, the constitutive expression of certain transgenes is sometimes useless, even harmful in normal conditions. Thanks to the present invention, it is therefore possible to express transgenes only under stress conditions.

In the context of the invention, the inventors have also discovered a novel protein, namely a new mannitol transporter in Apium graveolens celery, called AgMaT3. Therefore, the present application also relates to a peptide comprising a sequence which has at least 70% identity with the sequence SEQ ID No. 8, preferably at least 80% identity, or even 90% or even 95% of identity. identity with said sequence SEQ ID No. 8. Preferred peptides according to this aspect of the invention are peptides comprising or consisting of SEQ ID NO: 11. Other preferred peptides are those which are coded by all or part of the sequence SEQ ID No. 7, 13 or 14, or by a sequence resulting from the sequence SEQ ID No. 7, 13 or 14 because of the degeneracy of the genetic code.

Preferably, such an amino acid sequence according to this aspect of the invention has the ability to transport mannitol through a lipid bilayer, especially in plant cells.

The application also relates to the nucleic acid sequences coding for the peptides as described above. According to one possible implementation, said sequence is downstream of a promoter according to the invention, for example SEQ ID N ⁰ L

Legend of figures

Figure 1: Maps of cloning vectors used

1A: pDR Vectors 195 and 196

Multiple Site of Cloning of pDR 195: XhoI-NotI-SacIll-BamHI

Multiple Site of Cloning of pDR 196: SpeI-BamHI * -Smal-PstI-EcoRI-EcoRV-Hindlll-SalI-XhoI-

Acc65l-KpnI-Bam *

In bold are represented the enzymes which do not cut elsewhere in the plasmid and with *, those which cut twice in the SMC.

1B: Plasmid pBI 101-GUS-R1R2 (13942 bp) and Plasmid pBI 101-GFP5-R1R2 (13116bp)

Figure 2: Celery maintained for three weeks with water or fertilizer solution (control plant) and with 300 mM NaCl (stressed plant).

Figure 3: Northern hybridization of storage parenchyma, xylem and phloem mRNA of petioles of "control" or "stressed" plants with NaCl (300 mM, 3 weeks) by probes produced from the sequences obtained by the subtractive library. Quantification was performed using the 26S ribosomal probe. Abbreviations: PaT, Parenchyma Witness; PaN, Parenchyma NaCl; XT, Xylem Control; XN, Xylem NaCl; PhT, Phloem Witness; PhN, Phloem NaCl.

Figure 4: Graphical representation of the level of expression of the probes tested during hybridization in Northern, depending on the tissue. Abbreviations: AgMaT3, Apium graveolens Mannitol Transporter 3; AgMT2 and 3, Apium graveolens metallothioneins 2 and 3.

Figure 5: Absorption of mannitol (0.55 mM) by RS453 yeasts complemented by dpDR (A), or by AgMaTMpDR (+), or by three independent AgMaT3 / pDR clones (*, O and x), as a function of time. The kinetics performed with the strain transformed by the empty plasmid serves as a control. The values correspond to the mean ± standard error of the same experiment with three repetitions per point. Figure 6: Alignment of the nucleotide sequences of the dAgMaT cDNAs 1, 2 and 3. The sequences were aligned by the Clustal method of the MEGALIGN program (DNAstar, Madison, WI). Residues identical to the consensus sequence were highlighted. The ATG and stop codons are framed.

Figure 7: Alignment of the Protease Sequences of AgMaT 1, 2 and 3. The deduced amino acid sequences were aligned by the Clustal method of the MEGALIGN program (DNAstar, Madison, WI). Residues identical to the consensus sequence were highlighted. Framed residues correspond to the sequences held between the "sugar" carriers of the MFS family (according to NCBI Conserved Domain Search, www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). The surrounded methionine corresponds to the beginning of the initially cloned protein sequence.

Figure 8: Promoter and genomic sequence of AgMaT3 and translation of the coding region. The predicted peptide sequence is given using the one-letter abbreviation. The italicized residuals correspond to the conserved sequences of the MFS sugar subfamily subfamily. Putative transmembrane helices are underlined. The numbering is based on the translation initiation site.

Figure 9: Promoter sequence of AgMT2 and translation of the coding region. The predicted peptide sequence is given using the one-letter abbreviation. The numbering is based on the translation initiation site.

Figure 10: Promoter sequence of AgMT3 and translation of the coding region. The predicted peptide sequence is given using the one-letter abbreviation. The numbering is based on the translation initiation site.

Figure 11: Location of certain cis elements (identified by computer analysis) on the promoter regions of AgMaT3 and AgMT 2 and 3, used for constructions. The positions of the patterns are indicated from the translation initiation site. The elements placed on the complementary strand (-) are represented under the line.

Figure 12: Sequence of the 5 'upstream regions used as promoters. The sequences are represented until the first ATG corresponding to the site of initiation of the translation. The regions cloned upstream of the reporter genes are underlined and their length is indicated in brackets. SEQ ID NO: 1 (AgMaT3), SEQ ID NO: 2 (AgMT2) and SEQ ID NO: 3 (AgMT3) correspond to the underlined sequences, SEQ ID NO sequences ⁰ 4 (AgMaT3), SEQ ID NO: 5 ( AgMT2) and SEQ ID No. 6 (AgMTS) correspond to the sequences in bold; they end just before ATG's ¹ A '.

Figure 13: GUS staining of mature leaves of Arabidopsis plants expressing the following constructs: AgMaT3 r.uiad (A, B), AgMT2 :: uiad3 (C, D), AgMT3 :: uiad3 (E, F). Photos represent either the whole sheets (A, C, E, 5mm scale bar) or cross sections (B, D, F, 50μm scale bar). All photographs were taken from plants after salt stress.

Figure 14: Gus test results fragments root and leaf of the plants (Kemer tomatoes) transformed with the construct pAgMat3-GUS-tNOS a) one plate ^era plants 37-84 and controls; b) ^2nd Plate: plants 85 to 96 and controls. Plants subjected to salt stress (50mM for 4 days) are indicated in bold italic. The level of Gus staining is scored from 0 to 3. A GUS positive control under control of a constitutive promoter and a control of untransformed KEMER plants are used as controls.

Figure 15: Gus test results on stem fragments and leaves of six plants (Kemer tomatoes) transformed with the construct pAgMT2-GUS-tNOS. The level of Gus staining is scored from 0 to 3. A GUS positive control under control of a constitutive promoter and a control of untransformed KEMER plants are used as controls.

Figure 16: histo-cytological analysis of Gus positive plant fragments (Kemer tomatoes) transformed with the construct pAgMT2-GUS-tNOS and observation of staining in the vascular tissues. The reading took place 4 days after the test. The first observations show a blue color rather concentrated on the vascular tissues of the stem, in some cases, only on the veins of the leaves.

Figure 17: tissue sections of plants (Kemer tomatoes) transformed with the pAgMT2-GUS-tNOS construct. Tissue sections of the plant 13 at the stem (A) and plant 103 at the leaf (B) and the stem (C). These cytological data indicate an expression at the level of phloem tissues.

Experimental part

The team of Bohnert et al. (2001) conducted a large-scale genomic study of various organisms (Rice, Mesembryanthemum crystallinum L., Arabidopsis, Maize, Tobacco and Barley) in response to salt stress, on different tissues (mainly roots and leaves) at different stages of development and in very variable conditions (concentrations and duration). This study was pursued by insertional mutagenesis and QtL (guantitative trait ioci) studies, carried out on rice to identify the characters allowing this species to be tolerant of salt stress (Koyama et al., 2001). Global searches were also performed on the NaCl-treated Arabidopsis thaliana transcriptome (Kreps et al., 2002) and on the rice proteome (Salekdeh et al., 2002) in response to moderate salinization (100-150 mM). NaCl). These techniques, which study all the genes and proteins expressed in the plant during saline stress, are fully exploitable only when the entire genome of the species under study is available. is not the case of celery. The team of Masmoudi et al. (2001) searched for differential expression of wheat root cDNAs whose expression is modified by treatment with 200 mM NaCl. The technique used is much more targeted (to study the genes induced in a particular organ) but does not always identify many genes. The present inventors have therefore chosen to use the subtractive library technique which makes it possible both to clone the genes specifically induced by salt stress, and to obtain a large number of cDNAs, which are much more representative of the transcriptional state of the gene. plant, during this environmental constraint.

In addition, there is little or no information on the tissue localizations of the different genes involved in salt tolerance. The phloem, for its part, is a key tissue in the redistribution of Na ⁺ between different organs (Lohaus et al., 2000). It is also involved in the transport of osmolytes to the target accumulating tissues (Popova et al., 2003). Nyo-inositol and ABA are transported rapidly in the screened tubes, acting as down-stream signals (from the leaves to the roots) and activating many of the mechanisms necessary for the establishment of salt stress tolerance (Nelson ef a /., 1999). It is recognized that phloem plays an important role in the delivery of informative molecules to organs located at a distance (Ruiz-Medrano et al., 2001). It therefore seemed interesting to study the expression of genes during salt stress in this complex tissue. The inventors worked on a subtractive library constructed from phloem of plants having undergone salt stress and phloem of a control plant (in normal hydration).

The most suitable plant for this study was Celery, moderately sensitive to excessive macroelements or salt stress (De Pascale et al., 2003). This tolerance has been linked to an accumulation of mannitol (osmolyte) at the expense of sucrose by modification of enzymatic activities, mannose-6-phosphate reductase and mannitol dehydrogenase (Noiraud et al., 2000). Another benefit of celery is that phloem tissue (phloemian parenchyma cells + conductive complex) can be easily isolated, microsurgically, from the rest of the conductive bundle (Noiraud, 1999). It is therefore the variety Celery branch Apium graveolens L. Dulce (Green Elne cultivar) which was used by the inventors.

The objective is to identify gene promoters induced in the phloem by salt stress in Celery by the subtractive banking technique.

Example 1: Materials and Methods

I. Plant material

/. A. Celery branch

The Celery branch stems used are sown and grown on greenhouse potting soil. The plants are automatically watered for 90 sec every 3 hours during the day and receive a fertilizer solution (Peters solution 20:20:20 containing N, P and K at 100 ppm), three times a week. Once a week, spraying of the heart and young leaves should be done with nitrate calcium at 4 g. The ¹ alternating with Cosynol SC at 10 g. L ^"1 , to prevent rotting Celery (brown heart disease).

/. B. Salt stress (Celery)

When applying salt stress, two lots of Celery branch are prepared: "control" plants (T) and "stressed" plants (S). The 5.4 L jars are covered with a black plastic film to limit evaporation. Saline stress begins at a concentration of 25 mM NaCl, then increases in increments of 25 mM at each intake (twice 500 mL per pot per day, morning and evening) in water or fertilizer until concentration is reached. desired is 300 mM NaCl. The "control" plants are watered in the same way, but without adding salt. Salt stress is applied for three weeks. Fertilizers and calcium are applied with the same frequency as on the control plants.

/. C. Harvesting and preservation of tissues and organs

After 3 weeks of salt stress, the tissues are harvested according to a certain criterion (age or size) and preserved. One leaflet, the parenchyma, phloem, xylem of the petioles corresponding to the leaflet and the roots are taken from several plants and pooled according to the established criteria before being frozen in liquid nitrogen and stored at -80 ^° C.

/. D. Arabidopsis thaliana culture

The cress is used for plant transformation by the promoter sequences of the different genes studied. The plants of Arabidopsis thaliana ecotype Columbia (CoI-O) are sown and grown on potting soil in the greenhouse, under conditions of short days (day ≈ 21 ° C, 8h and night ≈ 17 ° C, 16h) for the development rosettes, then in long days (day ≈ 21 ° C, 16h and night≈ 17 ⁰ C, 8h) for the induction of flowering, until the rise of the flower stalk. The plants are automatically watered, in their saucers, for 90 sec every 3 hours during the day and receive a fertilizer solution (Peters solution 20:20:20 containing N, P and K at 100 ppm), three times a week.

II. Biological material for molecular approaches

//. A. Host microorganisms II. A. 1. Bacteria strain

The Escherichia coli DH5α bacterial strain (GibcoBRL) is used for cloning and amplification of DNA and PCR fragments. These bacteria are cultured at 37 ^° C., with solid or liquid dYT medium, in the presence of the appropriate antibiotic; ampicillin at 100 μg.mL ^"1 , kanamycin at 100 μg.mL ^{" 1} or gentamycin at 50 μg.mL ^"1. //. A. 2. Yeast strain

The RS453 yeast strain (Sauer and Stadler, 1993) is unable to grow on medium without uracil. The plasmid pDR carrying the URA3 gene is used to transform this strain. This strain is cultured with YPD medium, if it has not been supplemented, otherwise, on SC-glucose medium (without uracil). //. A. 3. Strain of Agrobacteria

The Agrobacterium strain used for the transformation of the floral stems of Arabidopsis ihaliana is Agrobacterium tumefaciens LBA 4404. It is cultured on YEB medium or in LB medium with stirring, at 28 ^° C., in the presence of rifampicin at 100 μg. mL ^"1 and streptomycin 200 μg.mL ^{" 1} .

//. B. Cloning Vectors

The maps of the cloning vectors used are given in FIG. 1. The vector used for the cloning of the PCR fragments is the plasmid pGEM-T-Easy (Promega).

The expression vectors pDR195 or pDR196 were used to complement the S. cerevisiae yeast RS453 strains. These vectors were used for the heterologous expression of plant mannitol transporters in yeast under the control of the PMA1 promoter.

The plasmid pDONR 207 (Invitrogen) was used as a donor vector in the kit "Gateway

Technology "(Invitrogen).

Plasmids pBI 101-GUS-R1 R2 and pBI 101-GFP-R1 R2 have the borders R1 and R2 (atfR1 and aftR2) which allow the cloning of the promoter of interest upstream of the GUS reporter genes (β-glucuronidase, uidA). or GFP (Green Fluorescent Protein 5-ER), by recombination between the input clone and the destination vectors pBI ("Gateway Technology" kit, Invitrogen). After the recombination step, the vector pBI 101 carries the reporter genes encoding GUS or GFP, under the control of the promoter studied, and the NOS terminator.

III. Methods

///. A. Extraction of Total RNAs from Plant Tissues

The technique used to extract total RNAs has been described by Kay et al. (1987).

///. B. DNA cloning by amplification (PCR)

III. B. 1. Amplification of genomic DNA by PCR

10 μg of DNA are placed in the presence of 50 μl of reaction medium containing 250 μM of dNTP; 1 μM sense and antisense primers; 1 U of Taq DNA polymerase "GoTaq" enzyme (Promega) and 1X of PCR buffer. Each cycle comprises (after a first denaturation step of 1 min at 94 ^° C. and followed by a last step of elongation of 5 min at 72 ^° C.): a denaturation step of 15 sec at 94 ^° C.

⁰ C; a hybridization step of 2 min at the appropriate temperature and an elongation step at 72 ^° C., the duration of which is proportional to the size of the fragment to be amplified (1000 bp.min ^-1 ). cloning in the vector pDONR207 are as follows:

For cloning the promoter part of AgMaT3:

Primer 5 'δ'-GGGGACAAGT TTGTACAAAA AAGCAGGCTG AACAGAAACAATTGTGGATG- 3'

3 'primer

5'-GGGGACCACT TTGTACAAGA AAGCTGGGTA ATGTTGAGAA ACAATGGTCG-3 '

For cloning the promoter part of AgMT2:

5 'primer

5'- GGGGACAAGT TTGTACAAAA AAGCAGGCTG ACCCACTATC AACAATGATC-3 '

3 'primer 5'- GGGGACCACT TTGTACMGA AAGCTGGGTA TAAGATCGTT GTGGACTCTG-3 '

For the cloning of the promoter part of AgMT3.

5 'primer

5'- GGGGACAAGT TTGTACAAAA AAGCAGGCTT CTTTATTCTG CAGCTAGAGC-3 '

3 'primer

5'- GGGGACCACT TTGTACAAGA AAGCTGGGTG CTTGAAGTAA GGTGGTATGC-3 '

The choice of the hybridization temperature depends on the preliminary calculation of the melting temperature (Tm).

The amplification products obtained are analyzed on 1% agarose gel.

///. B. 2. Amplification of DNA from reverse transcribed RNA (RT-PCR)

After denaturation of 6 μg of total RNA at 70 ^° C. for 10 min, the reverse transcription is carried out for 60 min at 42 ^° C. in the presence of 2.5 μM oligo (dT) ₁₈ primer, 500 μM dNTP and "M-MLV Reverse Transcriptase" (200U, Promega). The RNA / cDNA heteroduplexes obtained are denatured for 5 min at 100 ^° C. before a PCR amplification reaction of the target cDNA region is made by taking 2 μl of the first strand cDNA synthesis medium as a template. ///. B. 3. Cloning of the PCR fragments in the pGEM-T-Easy plasmid The vector of the "pGEM-T-Easy Vector Systems" system (Promega) is a linearized vector adapted to the direct cloning of the PCR products thanks to the grafted thymidine residues at each of its ends 3 '. The PCR products are ligated into 50 ng of plasmid pGEM-T-Easy in an insert / vector molar ratio of 3 overnight at 16 ° C in the presence of 3U T4 DNA ligase (Promega kit). 10 μL of the ligation medium were used to transform 200 μL of competent bacteria.

///. C. Transformation of competent Escherichia coli bacteria

The bacteria are made competent in the presence of CaCl ₂ .

///. C. 1. Preparation of competent bacteria

E. coli DH5α bacteria are made competent according to the method described by Sambrook et al,

(1989). The bacteria are then frozen in liquid nitrogen in aliquots of 200 μL and stored at -

8O ⁰ C.

///. C. 2. Transformation of thermocompetent bacteria

The transformation of competent E. coli DH5α bacteria is carried out according to the technique described by

Sambrook et al. (1989). The thermal shock occurs at 42 ° C for 90 sec. The addition of 800 μl of SOCt medium is followed by incubation at 37 ° C. with shaking for 1 h to allow the phenotypic expression of the antibiotic resistance gene carried by the plasmid. The transformation medium is then plated on a YT box containing the appropriate antibiotic. These boxes are placed at 37 ^° C. for 16 hours.

///. D. Plasmid DNA Analysis

III. D. 1. Minipréparation of plasmid DNA This extraction is based on the alkaline lysis technique of bacteria, adapted from Sambrook et al.

(1989); a bacterial colony is cultured in 5 ml of dYT medium containing the appropriate antibiotic overnight at 37 ° C with shaking. Cells from 3 mL of culture are concentrated by centrifugation for 1 min at 6000 g. The pellet is resuspended in 200 μL of solution 1

[25 mM Tris HCl (pH 8); 10 mM EDTA; 50 mM Glucose] supplemented with lysozyme at 1 mg.mL ^"1 final, then after vortexing, 400 μL of solution 2 [0.2 N NaOH, 1% SDS] and 300 μL of solution 3

[3M potassium acetate; glacial acetic acid 11.5% (v / v); pH 4.8] are successively added. After a 5 min incubation in ice, cell debris is sedimented by centrifugation for 10 min at 14000 g. The supernatant is transferred to a clean microtube where the

RNase A at 100 μg.mL ^'1 final is added before incubation at 65 ⁰ C for 15 min. The supernatant is purified by phenol / chloroform / isoamyl alcohol extraction (25: 24: 1). The DNA is precipitated with a volume of isopropanol, rinsed with 70% ethanol twice, dried and taken up in 20 μl of water.

///. D. 2. Establishment of restriction profile

One microliter of plasmid DNA solution (0.5 gL ^-1 ) was digested with various (3U) enzymes for 2 h at 37 ° C. The restriction products were analyzed on 1% agarose gel.

///. E. Sequencing and Clone Analysis

The most interesting clones are sequenced. The alignment of the nucleotide sequences, the search for restriction sites by the endonucleases, but also the search for open reading frames and the translation of the DNA fragments into amino acids were carried out with the software

Mac MoIIy Tetra (Soft Gene GmbH). The comparisons with the sequences contained in the databanks were realized thanks to the program BLAST on the server NCBI

(Http://www.ncbi.nlm.nih.gov).

The analysis of the promoters and the search for specific boxes of recognition and fixation of transcriptional regulatory proteins were carried out on the PLACE server (Higo et al., 1999, http: //www.dna.affrc.qo.ip /htdocs/PLACE/siqnalscan.htmiy

///. F. Subtractive Bank

This library represents all the genes whose expression is modified in a tissue following a given treatment. It was constructed according to the "SMART PCR cDNA Synthesis" and "PCR-Select cDNA Subtraction" kits (Clontech) from total phloem RNA extracted from either "control" plants or "300 mM NaCl stressed" plants. for three weeks. The cDNAs are directly produced and amplified from total RNA, before enzymatic digestion with Rsal, the cDNAs test (phloem of plants "stressed" S) are divided in two and on each sub-lot a different adapter is ligated giving S1 and S2. S1 and S2 are hybridized with the driver cDNA (phloem of "control" T plants) in excess, then combined and subjected to a second hybridization. The cDNAs corresponding to genes expressed under both stressed and control conditions will be able to hybridize. Only stressed plant cDNAs that have not hybridized with a "control" homolog and have different adapters can be amplified and cloned. ///. F. 1. Cloning the subtractive bank The PCR products (fragments of 90 to 900 bp) are directly cloned in the vector pGEM-T-Easy (Promega), then in the strain of E. coli DH5α. The colonies obtained are distributed on 96-well plates, thus cultured in liquid dYT medium and stored at -80 ^° C. (50% glycerol). ///. F. 2. Miniprocessing on a plate

The minipreparations are carried out directly on 96-well plates. The library is copied and cultured on deep well plates in 2 mL of dYT medium for 16 h at 37 ° C with shaking. After centrifugation (4000 g for 10 min at 4 ° C.), the pellet is resuspended with 100 μl of cold P1 solutions (Resuspension buffer, Qiagen) at 50 μg.mL-1 of RNase A, P2 (Lysis buffer, Qiagen), and P3 (Neutralization buffer, Qiagen) cold, stirring until the precipitates are formed After centrifugation, the supernatant is precipitated with one volume of isopropanol After re-centrifugation, the pellets are washed with 70% ethanol, then dried and resuspended in TE buffer (25μL, 50mM Tris, 10mM EDTA) with stirring The final concentration of plasmid DNA is determined spectrophotometrically.

///. F. 3. Filtration on filters

L ¹ plasmid DNA contained in each well of 96-well plates, is denatured with NaOH (final 0.4M) and deposited on nylon membranes Hybond-N (Amersham; 8 x 12 cm) using a replicator . The plasmid clones (100 ng) are deposited on the filters and fixed to the membranes by incubation at 80 ^° C. for 2 h. ///. F. 4. Hybridization and revelation of filters

The filters are placed in prehybridization for 4 h at 65 ^° C. (complex probe) or at 42 ^° C. (T7 probe), in a prehybridization solution [0.25 M NaP buffer, pH 7.2 (Na ₂ HPO ₄ , NaH ₂ PO ₄ ); 6.6% SDS, 1 mM EDTA, pH 8 and 1% bovine serum albumin].

The radioactive "complex probe" is produced by reverse transcription on total RNAs representing the genes expressed in a tissue following a given treatment, from anchored oligo-dT20. These probes will thus hybridize differentially on the clones deposited on the filters. The difference in intensity between the signals from the "stressed" and "control" complex probes makes it possible to identify the most interesting clones. The relative values obtained are readjusted to one another by calibration with a probe T7 internal to the deposited plasmid.

The first step of the reverse transcription reaction is denaturation at 70 ^° C. for 10 min, anchored RNAs and oligonucleotides, (dT) 20dA, (dT) 20dG, (dT) 20dC (2 μg each), mixed. The denatured forms are preserved by direct transfer in the ice. These RNAs are then diluted in a mixture preheated at 42 ^° C. for 5 min, composed of 1X RT buffer (50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl 2 and 10mM DTT), dNTPs. (0.8 mM each, except for dATP at 5 μM) and dATP-33P at 2.775 MBq in 7.5 μL (Amersham). Finally, 800 U of "MMLV reverse transcriptase" (Promega) are added to this PCR reaction and the mixture is incubated at 42 ^° C. for 1 h and at 70 ° C. for 15 min, then cooled in ice (denature the enzyme and retains linearization of cDNAs). So that there can be no interference with the hybridization of the probe, the template RNA is destroyed by adding 8 U Ribonuclease H (Promega) at 37 ⁰ C for 30 min. The probe is stored on ice while waiting for verification incorporation of dATP-33P. The elution is carried out with water in fractions of 200 μl whose radioactivity is estimated using a scintillation counter (Packard, TRI-CARB 1900 TR). The fractions containing the probe are pooled and are used as "complex probe" in order to hybridize the filters at 65 ^° C. overnight.

The quantity of plasmid DNA deposited at each spot is calibrated by hybridization with a T7 probe. This probe is produced from 100 ng of T7-oligo (20-mer) end-labeled (5 ') with γATP- ³³ P (1.85 MBq) fixed by reaction of "T4 polynucleotide kinase" (10U,

Promega) in 1X kinase buffer (Promega) at 37 ^° C. for 30 min, then blocked on ice. The filters are hybridized with the product of the reaction, overnight at 42 ^° C.

Hybridization of the filters is carried out for 16 h at 65 ^° C. (complex probe) or at 42 ^° C. T7 probe), in the presence of the radiolabelled probe diluted in the hybridization buffer (with the same composition as the prehybridization buffer). .

The filters are then washed twice for 10 min at 65 ^° C. or at 42 ^° C. in 2 × SSC; 0.1% SDS, 10 min at 65 ^° C. or at 42 ^° C. in 1 × SSC; 0.1% SDS, 5 min at 65 ^° C. or at room temperature in

0.5X SSC; SDS 0.1%. (SSC1X: 0.15 M NaCl and 0.015 M sodium citrate).

The filters are packaged in plastic bags and placed on "low energy" screens

(Kodak, Amersham), for six days inside exhibition tapes. The signals present on the filters are thus quantified by the Phospholmager system (STORM 820, Molecular Dynamics,

Amersham) and expressed in relative units. The values obtained with the probe T7 (probe internal to each plasmid) are used to calculate the relative intensities of the signal of each spot.

///. G. Northern Hybridization

III. G. 1. Electrophoresis and RNA transfer

The technique used for agarose denaturing gel electrophoresis and the transfer of RNAs by capillarity onto a nylon membrane is that described in Noiraux et al, 2000. After the transfer, the

RNA are fixed on the membrane by incubation at 80 ^° C. for 2 h.

III. G. 2. Preparation of the probe

The radioactive probe is prepared from a DNA fragment of interest. After enzymatic digestion,

I ¹ DNA was separated 1% agarose gel (Sambrook et al., 1989) of plasmid insertion and column purified "DNA gel extraction kit" (Millipore). The DNA pellet is resuspended in water at a final concentration of 125 ng / μL.

The radioactive probe is carried out with the "Prime-a-gene Labeling System" kit (Promega), based on the hybridization of a mixture of hexanucleotides with the DNA fragment to be labeled, denatured. The hexanucleotides serve as primers for the Klenow fragment that synthesizes a complementary strand by incorporating α32P-dCTP. The synthesis of the probe is carried out at 25 ^° C. for 2 h in the presence of

25 ng of denatured matrix DNA and 1.51.106 Bq of α ³² P-dCTP (specific activity 220 TBq.mmol-1).

At the end of the reaction, unincorporated nucleotides are separated from the probe by passing the mixture on a Sephadex G-50 column. The fractions containing the probe are pooled, denatured at 95 ^° C. for 3 min and then stored in ice until use.

///. G. 3. Hybridization of Northern The membrane is prehybridized for 4 h at 65 ^° C. in the prehybridization solution. Hybridization is carried out for 16 h at 65 ^° C. in the presence of the radiolabelled homologous probe (hybridization

Celery / celery). The membrane is then washed twice for 15 min at 65 ° C in 2X SSC; 0.1% SDS, 30 min at 68 ^° C. in 1 × SSC; 0.1% SDS, 15 min at 68 ^° C. in 0.5X SSC; SDS 0.1%.

The membrane is packed in a plastic bag and placed on screens for 3 hours. The RNA deposits are calibrated with a Celery 26S ribosomal probe, labeled with α ³² P-dCTP.

///. H. RACE-PCR

The coding sequences as well as the 5 'and 3' untranslated portions are cloned by means of the "Marathon cDNA amplification kit" (Clontech) from total phloem RNA extracts either from control plants.

The primers used for these clonings are chosen according to the prescriptions of the kit.The PCR products are complementary DNAs and are directly cloned into the pGEM-T-Easy vector (Promega), then in the strain E. coli DH5α.

///. /. RT-PCR in real time

Real-time RT-PCRs were made from RT (reverse transcription) on which a

PCR is performed from primers (Invitrogen) and TaqMan probes (Applied Biosystems, U.K.).

These are selected from the 3 'untranslated regions of the coding sequences of the tested mannitol transporters. These regions having the least similarity between them, the signal obtained is specific to the expression of one of these carriers.

Quantitative RT-PCR was performed essentially as described in Wagner et al., 2001.

The specific primers of AgMaT3 are:

¹ - ATA CAG CGG GGA TTA TAG CTT TG-3 'and 5'-ATC CGC AGG TAC TCC AAA AAT TT -3', in order to amplify a fragment of 101 base pairs specific to the 3 'non-coding region. The amplified fragment is verified by sequencing. The FAWI-labeled probe is 6 FAM-TAC CCG GTA TAT TCA

CTC-MGB (synthesized by Applied Biosystems).

For the amplification of Ag26S (control gene), the primers used are:

5'- AGC CGC TGG ACC CTA CCT-3 'and 5'-AGT TAT CTT TTC TGT TTA ACA GCC T-3', while the fluorescent probe is 6 FAM-CTA AGC CGT TTC CAG G-MGB.

Primers and probes are determined using Primer Express software, v.1.0 (Applied

Biosystems).

The fragments are obtained by PCR in the presence of "TaqMan Universal PCR Master Mix" 1X

(Applied Biosystems, Roche), 5 'and 3' primers at 0.9 μM each, 0.2 μM TaqMan probe and 1/300 ^th diluted RT product (RT produced from 10 μg d Total RNA). The fluorescence emitted by the TaqMan probes is detected thanks to the ABI PRISM 7700 detection system (Applied

Biosystems). Signals are quantified using Celery's 26S ribosomal primers and probes.

This gives Ct values defined as the number of the cycle at which the ΔRn value (normalized fluorescence intensity of the reporter dye) exceeds a threshold value generated by the software. Ct is inversely proportional to the concentration of the target sequence.

///. J. Cloning of Promoters and Genomic Sequences

The genomic sequences (introns + exons) and the promoters of known sequences are obtained by a technique based on PCR, by avoiding all the screening steps (bank phagic) traditionally used. The genomic DNA is divided into four lots, each being digested by a different enzyme. Selected enzymes give free-cutting cuts. Depending on the location of the restriction sites, the fragments obtained will be of variable size. Adapters of known sequences are then ligated to the ends of the digestion fragments. On each of the 4 lots, a PCR is carried out with a specific primer of the studied gene (GSP 1) and an adapter-specific primer (AP1). The second PCR or "nested" is practiced on the product of the first, with primers (GSP2 and AP2) more internal than the first ones, in order to optimize the specificity of the result. Up to 4 PCR fragments of variable size are thus obtained. The longest, carrying the most information, is purified. ///. J. 1, Extraction of genomic DNA

The tissues (young leaflets) are ground into a fine powder in a mortar in the presence of liquid nitrogen. Fifteen milliliters of 2X CTAB buffer [CTAB 2% (w / v); NaCl, 1.4 M; 20 mM EDTA; 100 mM Tris (pH 8); β-mercaptoethanol 0.2% (v / v)] preheated to 60 ^° C. are added for 3 to 5 g of ground powder and incubated for 30 min at 60 ^° C. The proteins and cell debris are removed by chloroform extraction. isoamyl alcohol [24: 1 (v / v)] followed by precipitation with cold isopropanol (0.5 volume) and at -20 ^° C. for 30 min. If the DNA filaments are visible, it is then possible to catch them and wash them with 76% ethanol containing final 10 mM acetate-NH ₄ for 20 min. Otherwise, centrifuge for 5 min at 5000 g and wash the pellet in the same way. The gDNA is again centrifuged and the pellet is air-dried and then taken up in 1 ml of TE buffer [50 mM Tris; 10 mM EDTA] supplemented with RNase A at 10 μg.mL ^-1 final and incubates for 30 min at 37 ^° C. The gDNA is precipitated by successive addition of 1.5 mL of TE buffer, 0.5 mL of NaCl (5 M) and 2 mL of cold isopropanol After centrifugation at 5000 g for 10 min, the pellet is washed with 70% ethanol, dried in air and taken up in TE buffer (50 μL).

The amounts of DNA are estimated by a spectrophotometric assay between 210 and 310 nm. An absorbance of 1 at 260 nm corresponds to a DNA concentration of 50 μg.mL-1. ///. J. 2. Construction of the genomic library

The genomic library is constructed according to the "Universal Genome Walker kit" kit (Clontech) from genomic DNA. The primers used are chosen according to the prescriptions of the kit. The PCR products obtained are directly cloned in the vector pGEM-T-Easy, then in the strain of E. coli DH5α for mini preparation and sequencing.

///. K. Transformation of Arabidopsis thaliana with a Celery Promoter Upstream of a Reporter Gene

III. K. 1. Gateway technology

The expression profile of the promoters was followed by transfer of their sequence upstream of a reporter gene. The use of gateway technology allows the passage of a DNA fragment

(complementary or genomic), from its PCR product, in a "donor vector" (pDONR

207) then in a "destination vector" (pBI-GUS-R1R2 or pBI-GFP-R1R2).

The primers produced (Invitrogen) for cloning these promoters contain the attB sequences, and are selected according to the data of the "Gateway Technology" kit (Invitrogen). PCRs have been performed on genomic DNA obtained according to § III. J. 1. After each ligation, the plasmids obtained are amplified in the E. coli strain. coli DH5α. The plasmids used contain the ccdB gene: in most bacteria, it is lethal. Thus, only the plasmids having carried out the recombination reaction (insertion of the DNA fragment and excision of the ccdB gene) can be amplified in the bacterium.

///. K. 2. Transformation of electrocompetent Agrobacteria

III. K. 2. a. Preparation of competent Agrobacteria

Preparation of Agrobacteria tumefaciens LBA 4404 is made according to Koncz and Schell (1986) to prepare 40 .mu.l aliquots (concentrations .3.1O ¹⁰ cellules.mL ^"1) which are stored at -80 ⁰ C until to use.

///. K. 2. b. Transformation of Agrobacteria by electroporation

The electroporation vats are placed at 4 ° C in ice. An aliquot of agrobacteria (40 .mu.l) is thawed and 2 .mu.l of plasmid DNA solution are added, all of which is deposited in the vats. The agrobacteria undergo an electric shock of 5 msec at 15 kV.cm- ¹ . Immediately, 960 μl of YEB medium are added, then the suspension is transferred and incubated at 28 ° C. with stirring for 1 hour to allow the phenotypic expression of the An antibiotic resistance gene carried by the plasmid The transformation medium is then spread on a YEB dish containing the appropriate antibiotics, placed at 28 ^° C. for 16 hrs, K. 3. Transformation of Arabidopsis thaliana

The transformation of Arabidopsis is according to the method described by Clough and Bent (1998), the transformed agrobacteria being cultured to an absorbance of 0.8 to 600 nm and then used to transform the floral stalks of Arabidopsis thaliana.

III. L. Study of the heterologous functional expression of plant transporters in the yeast Saccharomyces cerevisiae

III. L 1. Cloning of cDNA into the pDR expression vector

The pDR vector has the PMAI promoter and the ADH terminator which allow the expression of transporters in yeast. The cDNA sequence is extracted from the amplification vector (pGEM-T-Easy), by enzymatic digestion, with the same restriction enzymes (3U, 2h at 37 ^° C.) which cut the vector pDR, in order to perform cloning. oriented. The products of the digestions are controlled and purified on agarose gel 1%. The digested pDR plasmid is then dephosphorylated by the enzyme Shrimp Alkaline Phosphatase (4U, Promega) for 1 h at 37 ^° C., preventing its recircularization. Different enzymes are inhibited after each reaction, for 15 min at 65 ⁰ C. Ligation of cDNA I ¹ of the carrier in the PDR is then possible with the T4-DNA ligase (3U, Promega) overnight at 16 ⁰ C, according to an insert / vector ratio of 3. The prepared vector is amplified in the E. coli strain. DH5α coli before yeast transformation.

The preparation of competent yeasts and their transformation is done according to the protocol of Dohmen et al. (1991) ///. L. 2. Absorption of polyols by transformed yeasts

III. L 2. a. Yeast preparation for absorption The yeasts are prepared according to the method described by Noiraud et al. (2001)

///. L. 2. b. Absorption of yeast radiolabeled mannitol and sorbitol The uptake of radiolabelled polyols, manitol and sorbitols by yeasts is measured according to the method described by Noiraud et al. (2001), 100 μL of yeast suspension are incubated at 28 ° C in the presence of 100 μL of a solution containing 1.1 mWI of mannitol (0.55 mM final) with 5.8 KBq of mannitol [2-3H] ( specific activity 6.3.108 kBq.mmMM). For the sorbitol absorption test, the 100 μL of yeast suspension contains 1.1 mM of total sorbitol (0.55 mM final) with 16.6 KBq of sorbitol- ¹⁴ C (activity 1, 02.10 ⁷ kBq.mmol). ^"1 ).

///. L 2. c. Yeast Protein Assay

500 μl of cells prepared for absorption, washed and resuspended in MES without uracil SC are placed in a test tube and supplemented to 2 ml with water. 500 μl of 15% NaOH are added, and the whole is boiled for 5 minutes. After cooling, 175 μL of HCl 11, 75 N are added to neutralize the solution and the mixture is vortexed.

The proteins are assayed according to the technique of Bearden (1978), based on the color change of Coomassie blue when it binds to a protein.

Example 2

I. Salt stress and Celery

In order to constitute a tissue reserve for the construction of the subtractive library and the production of complex probes, Celery has been subjected to salt stress, by modifying certain conditions compared to treatments previously carried out (Noiraud, 1999). In order to standardize organ harvesting conditions, it is the actual age of the leaves (from the date of emergence) and not their general appearance (eg size) that is taken into account. Stress has a negative effect on leaf growth and development: a leaf under stress and an untreated leaf may look the same without being the same age.

Of the nine plants in each batch ("controls" and "stressed"), six are used for tissue harvesting and RNA extraction for the production of banks and probes. Only the leaf petiole phloem having reached an age of 3 weeks was used for the construction of the subtractive bank. These leaves have done all their development under stress conditions. General aspect of salt stress on plants

After a 3-week treatment with 300 mM NaCl, the plants are affected in their growth. The observation of the general appearance of Celery (fig.2) shows a difference in size and volume between the "stressed" and "control" feet, in correlation with the decrease in growth rate and the absence of emergence. leaves in plants treated with NaCl:

During salt stress, Celery shows a slowdown in growth and the emergence of new leaves. Inhibition of growth is a strategy of stress tolerance. This judgment is reversible because if the plants, after 3 weeks of salt stress, are sprayed with a solution containing no NaCl, very quickly the growth and the speed of emergence of the leaves is restored to a level identical to that noted before the salt stress. The outermost (oldest) leaves become senescent more rapidly, which may indicate a strategy for removing toxic ions from the youngest organs by accumulating and sequestering them in senescent organs. This sequestration in old leaves represents a salty stress tolerance strategy. In Celery, it is important to preserve the vegetative meristem intact so that it becomes inflorescential.

This implemented strategy must be coordinated at the level of the whole plant. This is possible by starting a new gene expression program. It is these genes specifically induced by this type of stress, which have been characterized by the inventors. This study focused on the phloem which plays a major role in the inter-organ exchanges of nutritive molecules and information molecules.

II. Realization of a subtractive bank

To identify genes specifically induced by salt stress, a subtractive library was performed using the Clontech PCR-Select cDNA Subtraction kit that identifies genes whose expression is stimulated in a given physiological condition (in this case, the salt stress). In theory, only these genes are obtained at the end of the analysis. The "stressed" plant cDNAs are subtracted from the cDNAs corresponding to the genes constitutively expressed in the normal state. PCR cycles can amplify specific salt stress genes.

As the amount of extracted phloem tissue is more limited than from leaves, an additional step of initial messenger amplification was introduced (Smart PCR cDNA synthesis kit, Clontech). The subtractive library was performed according to the provider's guidelines (Clontech PCR-Select cDNA Subtraction kit, Clontech). If a first phloem subtractive library was constructed and had good results in relation to a salt stress response (including heat shock proteins and a casein kinase 2 subunit), the amount of clones obtained was too low to build a bank.

A new phloem subtractive library has been performed, the results of which are detailed below: The average size of the inserts is between 90 and 900 bp. The clones obtained after subtraction and amplification are ligated into the pGEM-T-Easy vector (Promega). Colonies (the largest, 736 among about 750) are grown individually on 96-well plates. Replication was carried out for preservation after freezing at -80 ^° C., another replica was cultured in order to extract the plasmid DNA by minipréparation. //. A. Development of the screening system for subtractive banks

The number of clones obtained by the subtractive library is much too large (736 for the phloem subtractive library) to be able to analyze them independently of each other. In order to refine the search, the plasmid DNA of the clones obtained was deposited on filters that are hybridized with complex probes produced from RNA extracted from "stressed" or "control" tissues. The signals are then quantified and analyzed, and the so-called positive clones (according to the NaCl / control ratio) are retained. The radioelement chosen for the labeling of the probes is Phosphorus 33 with a concentration of 50 ng.μL ^-1 of plasmid DNA // B. Deposition and hybridizations of the clones obtained following the subtractive library Nitrocellulose filters (96 deposits) were made to identify the clones: the minipreparations are carried out in the plates on which the host bacteria have grown after replication of the frozen bank. Thus 736 independent clones could be deposited on membrane and analyzed. The membranes are hybridized with complex probes carried out by ³³ P labeling following reverse transcription on RNA extracted either from control phloem or from phloem of stressed plants. The radioactive markings obtained are counted (Phospholmager,) and compared. Thirty clones showed a strong signal under stress conditions and their plasmid DNA was sequenced (Table I).

Size Annotation Homology BLAST Number

(Pb) E-value accession functional

500 oxidoreductase, Arabidopsis NP_173786 5.1

Zn-binding dehydrogenase

300 no results - - -

90 metallothionein AgMT2 P. brachycarpa AAC62510 1e-04

440 chlorophyll a / b protein binding of CP29 Tomato CAA43590 3e-43

350 14-3-3 family protein Tomato P93211 3rd-58

310 no results - - -

800 NifU-like protein Arabidopsis NP 193953 1e-05 or dehydrin Barley AAD02257 0.001

800 beta-galactosidase Arabidopsis T00787 2e-35

700 hypersensitive-induced reaction protein Barley AAN17454 6th-44

320 Unknown Arabidopsis AAK31144 1e-05

150 no results - - -

600 chlorophyll a / b-binding protein Tomato S14305 6th-23rd

170 no results - - -

180 mannitol transporter AgMaT3 Celery AAG43998 2e-20

350 metallothionein AgMT3 Vine CAB85630 2nd-09

470 dnaK-type molecular chaperone hsc70 Tomato JC4786 2nd-76

660 high molecular weight heat shock protein Apple Tree AAF34134 5e-40

900 lipid transfer protein Arabidopsis NPJ77181 3rd-13

400 fiber annexin Cotton T31428 1e-58

800 RUB1 conjugating enzyme Tomato AAG23847 5th-54

900 cinnamyl alcohol dehydrogenase A. cordata P42495 2e-69

620 40S ribosomal protein S27 Arabidopsis NPJ91670 2e-33

600 60S ribosomal protein L30 Lupine O49884 1e-55

650 6OS ribosomal protein L28 Arabidopsis NP_194670 7th-41

450 no results - - -

250 no results - - -

250 no results - - -

640 no results - - -

Table I. cDNA strains obtained from the subtractive phloem library that showed a high stress / control ratio and differential hybridizations. The approximate size of these fragments is indicated in base pairs. The homology and the corresponding accession number were obtained by comparison with the BLAST databases. II. C. Results and bibliographic interpretation of positive clones in relation to stress and

Qhloème

Among the 30 positive clones, sequences could be identified which have similarities with genes whose role in stress has been documented (in particular heat shock proteins,

HSP, dehydrins and metallothioneins). Three sequences are identical and belong to the same gene and 8 others do not correspond to anything known. Of the remaining 21, 3 sequences are more particularly studied.

//. C. 1. AgMaTS, a third mannitol transporter in Celery?

The partial sequence found is very similar to the other 2 mannitol transporters identified in Celery: AgMaTI and AgMaT2 (Noiraud et al., 2001a). The isolated partial sequence in the phloem subtractive library is slightly different and has been named AgMaT3.

Transporters such as AgMaT3 could play an important role in salt stress tolerance by allowing the transport of osmoprotectants such as mannitol into the plant.

This transporter represents a good candidate in response to salt stress in the phloem.

//. C. 2. Metallothioneins

Four sequences correspond to metallothioneins (MT). Two are perfectly identical and belonged to the 3 'region of AgMT2 (Apium graveolens metallothionein 2). A third sequence is also homologous to AgMT2, but located upstream of the two previous ones. A fourth sequence also corresponds to a metallothionein but this is homologous to AgMT3. These names are attributed according to Vilaine et al. (2003).

Metallothionenins are small, low molecular weight, cysteine-rich, metal binding proteins (Zn, Cd) found in all animal and plant species and prokaryotes. They are also involved in stress responses such as metal ion treatments, heat shock (Hsieh et al., 1995), glucose and sucrose deficiency (Chevalier et al., 1995) or in cases of severe concentrations of it (Chatthai et al.,

1997), low temperatures (Reid and Ross, 1997), injury, and viral infection (Choi et al., 1996).

//. D. Complementary analysis of clones

The subtractive library analysis revealed a number of sequences induced by salt stress. If these libraries were made from phloem-derived RNA, this does not exclude that the identified genes are also expressed in other tissues. This is why the location of the sequences obtained is specified by northem reverse to validate their phloem specificity. The thirty clones presenting the most positive signals following the analysis of the filter bank are deposited on hybrid minifilters by new complex probes, produced from phloem or xylem extracted RNA from Celery leaf petioles "controls" or "stressed" (PhT and PhN or XT and XN, respectively). Ten clones were selected at the end of this analysis as specifically induced by salt stress, with some of them having a high phloem / xylem ratio (Table II).

Table II. CDNA sequences obtained after differential hybridizations from the subtractive phloem library with complex probes. The approximate size of these fragments is indicated in base pairs. Clones are classified according to the specificity of their expression during a salt stress in the phloem. Only clones with a ratio greater than 2 are inserted in this table. The ratio between phloem and xylem is calculated after hybridization with complex probes obtained from RNA extracted from phloem and xylem of salt-stressed plants.

The screens are standardized using a T7 probe (internal probe of the plasmid used for cloning the library). Among the clones of interest are the mannitol transporter AgMaT3 and the metallothionein MT2. Metallothionein MT3, whose signal in the control phloem is zero but whose Phloem / xylem ratio is high, is also found.

In order to refine these results, "Northern blots" were made by depositing RNA extracted from phloem, xylem and parenchyma storage of petioles of Celery "witnesses" or "stressed". The membranes are hybridized with radiolabelled probes made from the sequences selected for their tissue specificity (the inserts of the tested clones are extracted by digestion and purified before labeling). The results of these hybridizations are presented in FIG. 3. These hybridizations are quantified using a Celery 26S ribosomal probe and the intensities are measured by the revealing system (Phospholmager: STORM 820). The values obtained are reduced in percentage; the 100% for the expression of each probe in the "control" phloem (PhT), the comparisons are made from this tissue (FIG.

The AgMaT3 probe focused more specifically on the phloem "stressed" (PhN, 225%), confirming the previous results. The expression of AgMaT3 is also stimulated by salt stress in the storage parenchyma (PaN, 171%), and in the xylem. Northern results confirm that AgMaT3 is a gene whose expression is regulated by salt stress.

The 2 metallothioneins AgMT2 and AgMT3 are summer and react rather differently. AgMT3 is mainly expressed in phloem and this expression is very strongly stimulated following salt stress (224%). The stimulation of AgMT2 expression in the phloem following salt stress is less strong (133%). The expression of these 2 metallothioneins is mainly observed in the phloem in control and stressed plants (although it is also stimulated in the xylem following salt stress, the expression remains much lower than that measured in the phloem. therefore specific to phloem and their expression, especially for AgMT3, is indeed induced by salt stress // E. Conclusion

The aim of this first part was to identify genes whose expression is induced (or stimulated) during a salt stress in the celery phloem. The realization of the subtractive library required a development to obtain a maximum of clones: the final stage of amplification of the bank could be realized only by using a strain of bacteria different from that recommended by the supplier of the kit. Early analyzes of a small number of randomly selected clones showed that subtraction was effective. The large number of clones obtained led the inventors to look for a system making it possible to control the efficiency of the subtraction. The minifilters produced made it possible to test more than 700 clones for their ability to preferentially hybridize RNA from phloem of stressed plants to control plants. Signal normalization was performed by the T7 probe present on the cloning plasmid.

Clones reproducibly giving the most intense signals were sequenced and subjected to computer analysis (Table I). To refine these results and to verify if the identified sequences were indeed specific for phloem and salt stress, the 29 corresponding plasmids were redeposited on a new membrane and subjected to hybridizations with radiolabelled probes made from phloem of stressed or unstressed plants. but also xylem of the same plants, keeping only the sequences with specificity and induced by salt stress. A limited number of sequences were thus selected before a final check on Northern, including storage parenchyma as additional tissue. From these results, it is clear that both metallothioneins and the mannitol transporter AgMaT3 have the most interesting profiles.

The various results obtained led the inventors to continue the work by identifying promoters of the genes of interest, namely metallothioneins 2 and 3 and the new mannitol transporter AgMaT3.

The next step is to search for the promoter of these genes, using a genomic library and monitoring their activation in the various organs and tissues of the plant, during a salt stress.

III. Characterization of AgMaT3, a new celery mannitol transporter

Analysis of the phloem subtractive library made it possible to retain thirty clones presenting a strong signal under stress conditions on the 736 clones deposited on the membranes. Partial sequence (181 bp) was chosen because it corresponds to a mannitol transporter a compound qualified as

"compatible solute" in response to salt stress. Today, 2 mannitol transporters have been identified in celery: AgMaTI and AgMaT2 (Noiraud et al., 2001 a and b).

///. 1. RACE-PCR on the partial sequence of ApMaT3

RACE-PCR is performed on cDNAs produced from stressed phloem polyA RNA, to which adapters have been ligated, to amplify the 5 'and 3' moieties. These cDNA sequences

5 'and 3' are amplified by PCR between primers selected on the partial sequence of 181 bp (from the subtractive phloem library), and specific primers of the adapters according to the guidelines of the "Marathon cDNA Amplification" kit (Clontech). These primers are chosen so that the 5 'and 3' fragments can overlap and align to reconstitute the complete cDNA sequence. Once the sequences 5 'and 3' obtained, new primers were designed to amplify the entire cDNA AgMaT3 I ^1. (5 'primer;5' GAC TAG TCC CAA GAA TCT GAG TTC

ACC-3 'and 3' primer; 5'- CCG CTC GAG CAT CAC AAA GCT ATC ATC C-3 '

L ¹ cDNA AgMaT3 has a total length of 1796 bp. It contains an open reading frame of 1443 bp. This sequence is roughly the same size as the other two mannitol transporters (1766 bp for AgMaTI and 1781 bp for AgMa 72).

III. 2. Analysis of the Oseotide Sequence Derived from the AgMaT3 cDNA

The 1443 bp of the open reading frame of ¹ cDNA AgMaT3 encode a protein of 481 amino acids, shorter than those encoded by AgMaTI (513 aa) and AgMaT2 (524 aa). AgMaT3 shows 82

% and 73% similarity with AgMaTI and AgMaT2, respectively. This shorter size is actually due to a cloning artifact. Indeed, using new primers (primer 5 ': 5'-AGC TTC

GAC CAT TGT TTC TC-3 "and primer 3" 5'-CCG CTC CAT GAG CAT AAA GCT ATA ATC C-3 '), the authors of the present invention managed to clone a new cDNA, of greater length. The sequence corresponding to the longest protein was named AgMaT3, the sequence corresponding to the shortest protein being renamed AgMaT3 '.

III. 3. Analysis of the New Nucleotide Sequence of I ¹ AqMaT3 cDNA

Computer analysis of the genomic sequence of AgMaT3 revealed a new ATG

(giving an open reading frame of 1572 bp, SEQ ID No. 14). Taking this ATG as a starting point for translation (Figs 6 and 8) the encoded protein is longer (524 aa, SEQ ID NO: 8) than that obtained by RACE-PCR (481 aa). The alignment of the AgMaTI, AgMaT2 and

AgMaT3 proves that this result seems much more likely, with greater homogeneity in the length and alignment of these three sequences (Figure 6).

III. 4. Analysis of the New Peptide Sequence Derived from I ¹ AqMaT3 cDNA

The 1572 bp of the open reading frame of the AgMaT3 cDNA encodes a protein of 524 amino acids, of the same size as those encoded by AgMaT2 and slightly longer than AgMaTI (518 aa) (Fig. 7). The similarity between these three transporters is strong, but nuanced by different protein portions (fig.7, non-gray spaces where the amino acids do not find a homologue in any of the other two sequences studied), thus indicating that these mannitol transporters are distinct and especially in the N and C terminal parts. The AgMaT3 protein of 524 amino acids has a theoretical molecular mass of 56 670 Da. The isoelectric point of this protein was estimated at 8.3, a value close to those determined for AgMaTI and AgMaT2 (8.3 and 8.4, respectively).

///. 5. Functional characterization of the protein AαMaT3 Heterologous expression in yeast

Protein sequence analysis shows that AgMaT3 has strong homologies with the other two mannitol transporters. The AgMaT3 and AgMaT3 'cDNAs are separately cloned into the pDR 196 shuttle vector, and then serve to complement the RS453 strain. After transformation, the yeasts possessing the plasmid AgMa 73 / pDR are isolated on SC glucose medium for their prototrophy with uracil, the URA3 gene being carried by the plasmid.

To verify that mannitol transport was realized by AgMaT3, the kinetics of uptake of ^[3 H] - mannitol strain complemented by the plasmid containing cDNA or not AgMaT3 or AgMaT3 'was followed. Three independent clones are tested corresponding to 3 transformation events with AgMaT3 / pDR in yeast. Absorption of tritium labeled mannitol was monitored as a function of time (Fig. Yeasts expressing the plasmid AgMaT3 / pDR absorb much more mannitol than yeasts transformed with pDR. The absorption values at 3 min are very close to those noted for AgMaTI under the same measurement conditions. This result indicates that AgMaT3 is a mannitol transporter. It should be noted that the results obtained during the uptake of mannitol by the yeasts> 4g / Wa737pDR / RS453 indicate that this transporter is functional while the protein is deleted from about forty amino acids.

///. 6. Cloning of the genomic sequence AαMaT3

The cloning of the genomic sequences is carried out on the same principle as that of the promoters, with the "Universal Genome Walker" kit (Clontech) using primers oriented in the opposite direction to those which made it possible to sequence the promoter. The coding sequence of AgMaT3 is interspersed with two introns of 264 and 805 bp (Figure 8). The partial genomic sequence represents 2772 bp, from the ATG codon. The exons deduced from this genomic sequence correspond perfectly to the cDNA sequences except at the level of the initiation of the translation. Presence of an AATAAA polyadenylation site at position 2822 (or 179 bp after the stop codon) on the sense (+) strand of the genome sequence ά'AgMaT3 (Figure 8).

///. 7. Analysis of the expression of AαMaT3 in different tissues of Celery before or after a salt stress

This analysis is carried out by RT-PCR in real time on different tissues (xylem, phloem, leaves, roots and parenchyma) of plants treated or not with 300 mM NaCl for three weeks, in order to study the level of expression and their stimulation. in response to stress.

The values obtained with the AgMaT3 probe are normalized by the 26S ribosomal probe of

Celery. A difference of 3.42 Ct (see ΔΔ Ct in Table III) between phloem "control" and "stressed" indicates that it takes 3.42 additional cycles to obtain the signal in the phloem control of the same intensity only in the phloem "stressed"; Expression of AgMaT3 is greater in the phloem of NaCl-treated plants. In addition, the PCR cycles allowing exponential amplification, a difference of 3.42 Ct corresponds to ^{23 '42} , or 10.70 times more AgMaT3 transcripts in the phloem of "stressed" plants than "controls". This result confirms those previously obtained in the subtractive library and Northern (fig.3 and Table II). Similarly, a difference of 7.52 Ct (see ΔΔ Ct in Table III) between "control" and "stressed" roots indicates that it takes 7.52 additional cycles to obtain the signal in control roots of the same intensity as in the "stressed" roots.

Table III. Results obtained by RT-PCR in real time with the AgMaT3 probe, in different tissues (phloem, xylem, parenchyma, leaves, roots) of control plants or treated with NaCl (300 mM for 3 weeks). These results were corrected by the values of the standard 26S ribosomal probe from Celery. Ct is the number of cycles required to perceive a given intensity signal. Δ Ct is the difference between the means of the Ct (average of the repetitions for a probe analyzed in a given tissue) obtained for the AgMaT3 probe after subtraction of the Ct average obtained for the 26S probe. ΔΔ Ct is the comparison (difference between two Δ Ct) between the different tissues. The higher the value of Ct, the more cycles it took to get a signal, and the fewer transcripts there were.

///. 8. Conclusion onAαMaT3

The information obtained during the absorption of mannitol as a function of time as well as the alignments of the nucleotide and protein sequences make it possible to conclude that AgMaT3 encodes a third mannitol transporter in celery branch. Real-time and Northern RT-PCR studies confirm that AgMaT3 expression is specific for salt stress. This mannitol transporter would therefore play an important role in salt stress tolerance.

IV. Promoter search and heterologous expression in A. thaliana

To search for promoters specifically induced by salt stress in the phloem, it was desirable to have a powerful promoter cloning system (the screening steps of a genomic library are particularly heavy). In addition, a first attempt The search for a promoter of the AgMaTI gene, from a genomic library in a phage λ, had been unsuccessful (Cyril Maingourd, 2001). In the research perspective of several promoters, it became necessary to use a faster technique. This is why the technique of PCR amplification of promoter regions from DNA sequences (Universal GenomeWalker kit, clontech) was used. The search for promoter sequences in Celery was carried out on the selected genes: AgMaT3 encoding the new mannitol transporter and the two metallothionein genes

(AgMT 2 and AgMT 3).

IV. A. Alphaomale Bank and Cloning of Promoters

The genomic DNA of Celery is separated into four batches on which four different restriction enzymes, EcoRV, DraI, SspI and HpaI are made to act, leaving free-edged ends, specific adapters being ligated thereto. Four different fractions are thus obtained, on which PCRs are carried out with a primer specific for the sequence to be amplified (chosen close to the codon).

ATG translation start) and a specific primer of the adapter. The fragments obtained are of variable size with a particular profile at each digestion. Only the longest amplified fragments are retained for cloning and sequencing. Some refinements are needed to obtain this library for the choice of restriction enzymes (one of the enzymes recommended by the supplier not cutting Celery's DNA) and the material used. For each gene studied, a promoter region upstream of the ATG is cloned into an intermediate vector (pDONR 207) and then transferred into a binary expression vector (pBi-GUS-R1R2 and pBi-GFP-R1R2) by the system.

Gateway (Invitrogen). This allows the study of the expression of reporter genes (GUS or GFP) under the control of the promoters described, after transformation of Arabidopsis plants.

The constructions made were used to transform agrobacteria. After verifying the presence of plasmids in agrobacteria, Arabidopsis plants are transformed according to the technique described in the Materials and Methods section.

IV. B. Promoter of the gene encoding the mannitol transporter AαMaT3

The ά'AgMaT3 promoter represents 589 bp upstream of the ATG (FIG 8, SEQ ID No. 4). The promoter regions ά 'AgMaT 1 and AgMaT 2 were also cloned and made 844 and 383 bp upstream of the ATG. These three sequences contain numerous A / T repeats characteristic of the promoter regions.

Transcription factor binding sites in response to different stimuli are searched using the PLACE software (http://www.dna.affrc.go.jp/sigscan/). Only the cis elements regulated by the sugar signal and the light are analyzed. The position of the cassettes is indicated relative to the site of initiation of the translation (ATG codon). The regulatory proteins can bind on the sense strand, noted (+) or on its complementary, antisense strand, noted (-).

IV. B. 1. Reply to the light

The GATA box (GATABOX, S000039), required for regulation by light (Teakle et al., 2002) has been identified 7 times in the AgMaT3 promoter (in -299 (-), -289 (-), - 269 (+), -264 (+), -254 (-), -

154 (+) and -77 (+)). GT-1 Link Site (GT1 CONSENSUS, S000198) (Terzaghi and Cashmore,

1995) is present 5 times upstream of AgMaT3 (-392 (-), -291 (-), -264 (+), -201 (-) and -154 (+)). The GATAA sequence (I-BOX, S000199), highly conserved upstream of light-regulated genes as well well in mono- and dicotyledons (Terzaghi and Cashmore, 1995), is found 3 times upstream of AgMaT3 (in -290 (-), -264 (+) and -154 (+)).

The "Tbox" (TBOXATGAPB, S000383, ACTTTG), involved in gene activation by light, was found in the AgMaT3 promoter at -455 (-). The transcription in response to the light of the tobacco psaDb gene depends on the Inr element ("initiator" INRNTPSADB, S000395, C / TTCANT (C / T) ₂ , element present 2 times upstream of AgMaT3 (-387 (+ ) and -189 (-)).

IV. B. 2. Answer to sugars

Sugars are now considered as molecules capable of regulating the expression of many genes.

The PYRIMIDINEBOXOSRAMY1A (CCTTTT, S000259) box, of response to gibberellin and also implicated in repression by sugars (Morita et al., 1998) is present in the promoter sequence 6'AgMaT3, twice (-398 (+) and -219 (-)). The TATCCA element

(TATCCAOSAMY, S000403), MYB protein binding site (Lu et al., 2002) is present only once in the AgMaT3 promoter (-299 (+)). The "W-box" element (WBOXHVISO1, S000442, TGACT), SUSI BA2 transcription factor binding site (sugar sjgnaling in barley, sugar inducible), is identified three times in the AgMaT3 promoter (- 419 (-), -367 (-) and -74 (-)). These elements suggest a regulation by sugars (glucose, sucrose).

IV. C. Promoters of AαMT2 and 3

Metallothioneins have been found both in phloem and salt stress, but also in aphids and viruses (Fanchon Divol, 2003). The results of the present salt stress analysis showed that the metallothioneins AgMT2 and AgMT3 are specifically expressed in response to this stress (Figure 3).

The promoter regions represent 1319 and 648 bp and were cloned upstream of the reporter genes (GUS and GFP) of the pBi vectors for AgMT2 and AgMT3 (Figures 9 and 10).

IV. D. Analysis of stress response cassettes

The computer analysis of the promoters revealed many potential cis-elements related to abiotic stress.

IV. D. 1. Response to abiotic stress

Abscisic acid (ABA) is present in all higher plants. This phytohormone is involved in several seed development events and regulates the expression of many genes in response to environmental stresses such as dehydration, salt and cold

(Busk and Pages, 1998). The analysis of AgMaT3, AgMT2 and AgMT3 promoters revealed ABRE (ABA-responsive element) c / s-regulatory elements, on which many ABFs (ABRE binding factor, members of a subfamily of bZIP proteins) were found. ) bind in response to a dependent ABA signaling pathway (Kang et al., 2002) to induce the expression of a large number of genes (Choi et al., 2000). In Arabidopsis, induction of the rd22 gene by ABA and dehydration involves MYC and MYB transcription factors (Abe et al., 2003 and 1997) binding to specific sites CANNTG (MYCCONSENSUAT, S000407) or C / TAACG / TG (MYB2CONSENSUSAT,

S000409), A / TAACCA (MYB1AT, S000408). The CANNTG sequence (S000407) is also the ICE binding site (inducer of JC3F expression) that regulates the transcription of genes encoding a CBF, in response to cold (Chinnusamy et al., 2003). Site S000407 is present upstream of AgMaT3 (-550

(+/-), fig. 8 and 11) and AgMT2 (-369 (+/-) and -158 (+/-), Figs 9 and 11). This site has not been identified in the AgMT3 promoter (Figures 10 and 11). The S000409 sequence was analyzed upstream of the AgMT2 gene at -820 (+), AgMT3 at -575 (+). S000408 is present in the Ag MT2 promoter at -642. An Arabidopsis MYB transcription factor induced by dehydration and salt stress (Urao et al., 1993) recognizes and binds to the TAACTG consensus sequence (MYB2AT, S000177). This was characterized upstream of AgMT3 in -575 (+).

The sequence CNGTTG / A (MYBCORE, S000176) represents the binding site of MYB which is also induced by water stress in A. thaliana. This sequence is identified in the AgMT2 promoters at -820 (-) and AgMT3 (-575 (-) and -563 (-)). The thermoregulated expression of a hs (heat shock) soy gene (Rieping and Schoffl, 1992) is regulated via a HSE element of the promoter (heat shock element), but this activity requires additional sequences: CCAAT boxes

(CCAATBOX1, S000030). These are present upstream of the AgMaT3 gene (-176 (+)). This box is not present upstream of AgMT3 whereas it has been identified six times in the AgMT2 promoter in -1280 (-), -834 (-), -1263 (-), -693 (- ), -1057 (-) and -624 (+).

IV. D. 2. Response to biotic stresses

Many signaling pathways in response to biotic and abiotic stresses are intertwined. Transcription factor binding elements in response to pathogenic attacks and injury were also selected as potential candidates for salt stress induction. A few of them have been listed when their motifs have been detected on AgMaT3, AgMT2 and AgMT 3 promoters.

Expression of many defense genes are regulated via cis elements such as the GCC box (GCCCORE, GCCGCC, S000430), identified at -284 (-) upstream of AgMaT3.

The AG motif (AGATCCAA, AGMOTI FNTMYB2, S000444) is a sufficient element to confer a response to the injury and following an elicitor treatment (Sugimoto et al., 2003). The S000444 motif was found in the AgMT3 promoter at -452.

The most important elements in response to biotic stress are W-boxes, WRKY protein binding sites (superfamily of transcription factors, involved in the regulation of variable physiological programs such as pathogen defense, senescence and trichome development). (Eulgem et al., 2000)). They bind to TGAC palindromic sequence boxes such as the "WB box" (WBBOXPCWRKY1, TTTGACT, S000310), present upstream of AgMaT3 at -74 (-) and AgMT2 at -169 (+).

The Arabidopsis NPR1 gene, a positive regulator of inducible disease resistance, contains in its promoter the "W-box" sequence (WBOXATNPR1, TTGAC, S000390), a WRKY binding site induced by a pathogenic infection or a treatment. salicylic acid (Yu et al.,

2001). This was identified three times in the AgMaT3 promoters (-418 (-), -366 (-) and -73 (-)), AgM72 in -168 (+) and Ag / W73 in -496. These elements were placed according to their location on the AgMaT3 promoter regions of AgMT2 and AgMT3 (Figure 11).

The promoter sequence of AgMT2 has few MYB binding sites, MYC or in response to I ¹ ABA but not less than five HSE elements on the strand (-) and one on the strand (+), as well as an element LTRE . The expression of these promoters upstream of a reporter gene in the tissues and organs of the plant is therefore interesting to follow during salt stress.

IV. E. Functional Analysis of the Promoter Regions by Heterologous Expression in A. thaliana Each sequence identified and cloned in Celery was placed upstream of two reporter genes GUS and GFP, in order to follow their expression in A. thaliana. Cloning was performed using gateway technology (Invitrogen) between the right and left borders of a pBI vector. The regions upstream of the ATG that have been cloned for each of the genes are shown in FIG. 12. The lengths of the cloned regions are 548 (AgMaT3), 649 (AgMT3) and 1319 base pairs (AgMT2). All cloned sequences include the putative transcription initiation site (located at position 541 for AgMaT3, position 1502 for AgMT2 and position 639 for AgMT3). The effective transformation of the plants is doubly verified, by germination in the presence of kanamycin and by PCR control with NPT-II specific primers. For all the experiments, two controls are added: a non-transformed CoHO line and plants transformed by the uiad gene under the control of the AtPP2-1 promoter (Dinant et al, 2003), which gives a strong signal in the phloem. The experiments are repeated on 5 plants for each promoter construct. The survival of the plants is tested after treatment with NaCl at concentrations between 50 and 250 mM. The ideal concentration to induce a salt stress response, without being lethal to the plant, nor to prevent the development of a flowering stalk and seed production, was determined at 100 mM.

The response is followed by a visual inspection of leaf color before and after salt stress and cross section for phloem color verification. The general observation of the leaves of the control plants (not subject to stress) does not reveal, after GUS staining, any special staining except in the case of AtPP2-1 and AgMT2. In all cases, the staining is limited to the veins. For AtPP2-1 and AgMT2, all veins are positively stained (type I to IV, Fig. 13C) whereas in the case of AgMaT3 and AgMT3, only major veins (type I) are stained (Fig. 13A and E) . In all cases, the veins in the petioles are also colored. In addition, the young and mature leaves are stained identically.

To determine the exact location of GUS expression in vascular bundles, cross-sections are performed on frozen material. For plants showing strong staining (AtPP2-1 and AgMT2 Fig. 13D), blue staining is evident in phloem cells but also sometimes in surrounding cells. This may be due to the diffusion of the staining, since it has already been shown that GUS staining is limited to phloem cells in the case of AtPP2-1 (Dinant et al 2003). For the AgMaT3 (Figure 13B) and AgMT3 (Figure 13F) constructs, staining is also evident in phloem cells, thus confirming the initial identification of corresponding cDNAs as specific to phloem cells. Staining is also detected from parenchymal xylem cells. Among these cells are probably vessel-associated cells (VACs), which are considered to be very similar in function to comparable phloem cells (Fromard et al, 1995). Salt stress induction may be either specific to the present test or part of a more general stress response of the plant. To test this hypothesis, groups of 5 plants from the same transformants were subjected to osmotic stress (no watering for 4 days), cold stress (4 days at 4 ⁰ C), stress at heat (4 days at 30 ° C) and stress injury (leaves pinched with a forceps 3 times in a row, taking care to avoid major veins) The plants were harvested the day after the stress. of 5 independent plants was used as control.The plants are colored as for salt stress.All plants (except for water and salt stress) are watered twice a day with 1OmL of tap water or a solution of fertilizer (once during the stress period) The leaves (fully mature or young shoots) are sampled at the end of the stress period and used for histochemical staining GUS (Jefferson et al, 1987). leaves are being discharged in liquid nitrogen and stored at -80 ^° C until fluorine GUS tests (Jefferson et al, 1987).

For AgMT2 plants, it was difficult to observe a difference between the different stresses, since the staining is already particularly intense in the control plants. However, no difference was observed in the staining pattern. In the case of AgMaT3, all forms of stress give similar staining patterns, with the exception of water stress and injury stress where no signal has been detected, although the plants are still healthy. In AgMT3 plants, GUS staining is evident in all forms of stress with the exception of injury stress.

Injury stress responses would use a different signaling pathway and do not lead to activation of the genes being studied.

The results are very similar to those obtained with celery. Both the 5 'region of AgMT3 and that of AgMaT3 lead to expression of the reporter gene in phloem (and to some extent also in xylem), but only in stressed plants and not in control plants. In the case of AgMT2, an important expression was observed in the phloem of transformed Arabidopsis plants, even under control conditions. An interesting point is the high level of expression of the fusion between the AgMT2 promoter and GUS in Arabidopsis in all the veins of the leaf (Fig. 13C), indicating that the expression pattern is phloem specific.

Example 3 Brassica Transformation

Brassica Transformation Protocol: Derivative of Brasileiro et al, 1992 and Bethomieu (Doctoral thesis: Obtaining genetically engineered transgenic cabbages by genetic transformation with a gene encoding a Bacillus thuringiensis endotoxin).

1. Seeding and Germination

The seeds are disinfected for 25 minutes with stirring in a solution of bayrochlore (3 tablets / liter of demineralized water) before being rinsed with sterile water and then dried on a blotter.

Sterilized, they are sown in tubes on an M1 germination medium (4.4 g / l of MS 5519 (Sigma), 20g / l of sucrose and 8g / l of agar (pH 5.8)). The seeds are cultured for 15 days at 20 ^° C. (16h photoperiod) to obtain seedlings at the 2-3-leaf stage.

2. Culture of bacteria

Two Agrobacteria are used: C 58pMP90 contains the plasmid pSCV with the T-DNA comprising the reporter gene expression cassette (GUS gene), under the control of the promoter sequences of the present invention (Kanamycin selection: 50 μg / ml; Rifampicin: 20 μg / mL), the second containing the tumorigenic t-DNA (pTl82-139) (Kanamycin selection: 50 μg / mL, Rifampycin: 50 μg / mL and Gentamycin: 20 μg / mL)

The bacteria are pre-cultured in 20ml of LB medium (Luria Bertani from Difco) containing the selection antibiotics plus 1 ml of glycerol-stock of the bacteria. These pre-cultures are stirred (200 rpm) at 28 ° C. for 24 hours. The cultures are made by adding 1 ml of the preculture to 20 ml of LB medium containing the selection antibiotics. The cultures are stirred (200rpm) at 28 ° C overnight.

3. Preparation of bacteria

If the optical density at 600 nm of the two agrobacteria cultures after one night is not 1, they are centrifuged for 10 minutes at 4000 rpm. The bacterial pellet is taken up with a sufficient volume of the liquid medium M4 containing 0.88 g / l of MS 5519 and 10 g / l of sucrose (pH 5.8) in order to arrive at the desired OD. The 2 bacteria are mixed according to the ratio Bact 2 / Bact 1: 1/6

4. Inoculation of plants and transplanting

The seedlings are inoculated with a 20 cm DEMARTEL forceps, dipped in the bacterial mixture and used to injure the stem at three different points above the cotyledons. The seedlings inoculated are cultured at a temperature of 20 ⁰ C (photoperiod 16h) until tumors appear at the points of injury. The tumors are subcultured on an M2 medium containing 4.4 g / l of MS 5519, 30 g / l of sucrose, 8 g / l of agar-agar, 400 mg / l of antibiotic (Augmentin) (pH 5.8)

5. Regeneration of buds

About 1 month after transplanting the tumors, the buds appear. When they have a size of 3 to 4 cm, they are isolated and put in tubes on an M3 medium containing 4.4 g / l of MS 5519, 30 g / l of sucrose, 8 g / l of agar-agar (pH 5.8) 200mg / l antibiotic (Augmentin) (pH 5.8). The tumors are fragmented and transplanted on M2 medium to regenerate other buds. Plantlets are rooted 3-4 weeks after isolation of buds on M3 medium.

6. Acclimatization of plants

The seedlings are transferred into potting looses and maintained at 22 ° C and 80% relative humidity for one week. They are then conducted as plants derived from sowing (temperature 20 ° C and ambient hygrometry), potted in culture pots for the production of seeds. The siliques reach maturity in 5 months.

Example 4: transformation of tomatoes.

Tomato Transformation Protocol (Binary Transformation System):

Derived from Filatti et al, 1987. 7 / Sowing and germination

The seeds, placed in a square of mosquito net closed by staples, are disinfected in a solution of bayrochlore (2 to 3 pellets per liter of distilled water and a few drops of Tween) during 20 minutes. They are then rinsed three times for 15 minutes in sterile distilled water and then dried rapidly in blotting paper. The sterilized seeds are sown in jars on a T1 germination medium (MS 6899, Sigma) comprising 2.2 g / l of MS 6899, 2 ml / l of

Vitamins of Nitsh & Nitsh, 1965, 30g / l of sucrose (pH 5.9, adjusted with KOH), and 8g / l of agar-agar.

The boxes are left for 24 hours in the dark, in an oven at 26 ° C. Placed in the light, the cotyledons develop. They can be used 7 to 8 days after sowing.

2 / Preculture of explants on solid TT2

An explant is cut by cotyledon and then placed on the pre-culture T2 medium comprising 4.4 g / l of MS 6899 2ml / l of Nitsh & Nitsh Vitamins, 0.9 mg / l of Thiamine, 200 mg of potassium dihydrogen phosphate (KH 2 PO 4 ), 30 g / l sucrose (pH 5.9, adjusted with KOH), 8 g / l agar-agar,

0.2mg / l 2,4-D (Sigma), 0.1 mg / l Kinetin (Sigma), 200μmol / l Acetosyringone (Aldrich). The underside of the explants is placed against the middle. The boxes are left in the light at 26 ° C for 24 hours.

3 / Preparation of bacteria:

Agrobacterium EHA 105 containing the plasmid pBI101 is used with the T-DNA comprising the selection gene expression cassette (kanamycin resistance gene) and the reporter gene (gene

Beta-glucuronidase Gus, under the control of the promoter sequences according to the present invention), (antibiotic Kanamycin: 50 μg / ml, Rifampicin: 50 μg / ml)

A preculture of bacteria is made in a 50ml Falcon tube, by seeding 1m! of glycerol-stock of the bacterium in 10 ml of LB or TP medium culture medium (Yeast-Tryptone of

Difco) containing the selection antibiotics. These pre-cultures are stirred (250 rpm) in the dark at 28 ° C. for 24 hours. The cultures are made by adding 1 ml of the preculture to 20 ml of LB or YT medium containing the selection antibiotics. The cultures are stirred

(250rpm), in the dark, at 28 ° C for the night. The next morning, the OD at 600 nm of one ml of bacterial culture is measured. If different from 2, cultures are centrifuged for 10mn to 3000

RPM. The supernatant is removed and the bacterial pellet resuspended with an adequate volume of medium

T2 liquid.

4 / Coculture:

At 28.5 ml of coculture medium (liquid medium T2 with acetosyringone alone, without ketinin or

2,4-D) per Petri dish, 2.5 ml of bacterial solution are added to the OD ₆ oonm 2.

The explants are taken from the preculture medium and added to the Petri dishes to incubate with the bacteria for 30 minutes. The explants are then dried on sterile blotting paper and then returned to the same solid T2 medium used for preculture. The boxes are placed in the dark, at 26 ° C for 48h.

5 / Washing the explants:

About 15OmL of liquid washing medium MS 6899 is placed in each sterile pot (single use) and soaked up to 50 explants (medium T3: 4.4g / L MS 6899, 2mL / L vitamins Nitsh & Nitsh, 30g / L of Sucrose and 400mg / L of Augmentin (pH 5.9 adjusted with KOH)). After about ten minutes of soaking, the explants are recovered using forceps and dried rapidly on sterile blotting paper.

6 / Transplantation of explants in regeneration environment:

The explants are transplanted on solid T3 medium cast in high petri dishes. Solid T3 medium:

4.4 g / l of MS 6899, 2 ml / 1 of Nitsh & Nitsh vitamins, 30 g / l of sucrose, 8 g / l of agar-agar, 2 mg / l of Zeatine (Sigma), 500 mg / l of Augmentin ( 600mg / l when using LBA4404 bacteria), 100mg / l of

Kanamycin (Sigma). After 2 weeks in the light at 26 ^° C., the explants are cut in half in order to differentiate the transformation events (the two ends of the same explant are considered as distinct) and to transplant them onto a new T3 medium. . The number of explants that regenerated 21 days after coculture is counted.

7 / Preparation of the development environment

The buds are separated from the primary explant 4 to 6 weeks after coculture and placed on T4 selective medium (2.2 g / l of MS 6899, 2 ml / l of Nitsh & Nitsh vitamins, 20 g / l of sucrose, 8 g / l). agar agar, 0.5mg / l Zeatin (Sigma), 300mg / l Augmentin (600mg / l when using the bacterium

LBA4404), 50mg / l Kanamycin (Sigma).

8 / Rooting transformed buds:

When the buds reach a few centimeters high, they are subcultured in large TPS pots on the rooting medium T5: 2.2 g / l of MS 6899, 2 ml / l of vitamins of Nitsh & Nitsh, 20g / l of

Sucrose, 2.5g / l Phytagel, 0.5mg / l AlA (Sigma), 300mg / l Augmentin (600mg / l when using LBA4404 bacteria), 50mg / l Kanamycin (Sigma). The plantlets synthesizing chlorophyll and developing roots are considered transformed. After determining the ploidy level of each seedling by flow cytometry or amyloplast counting, the diploid seedlings are acclimated and then planted in a greenhouse.

9 / Acclimatization - Greenhouse planting

The well-developed seedlings are rooted in 10 cm pots on Steckmedium. They are covered for two weeks with a plastic film in order to maintain a high hygrometry. The seedlings are then planted in 7-liter pots filled with Pouzzolan.

Salmon stress protocol development

Seedling of non-transgenic tomato seeds (Kemer) was carried out under conventional germination conditions: 10 seeds per plastic dish with blotting paper soaked with water and NaCl at different concentrations (H ² O, 50, 100, 150, 200 250 and 300 mM), two boxes per concentration.

The seeds were kept in the dark for 3 days and then exposed to light at a temperature of 25 ^° C. The observations were made 5 to 6 days after germination.

The H ² O control has 90% germination, the seedlings (cotyledon stage) measure about 2 cm.

Seeds receiving 50 mM NaCl have 45% germination and seedling size is

1 cm. Seeds receiving 100 mM NaCl and beyond did not show germination at the time of observation, and did not germinate 2 weeks later. The dose of 50 mM NaCl was therefore retained for the achievement of salt stress.

GUS test protocol

1 / Preparation of the reaction buffer

A solution A of 0.2M Na 2 HPO 4 (Merck, No. 238) 28.4 g / l is gradually mixed with a solution of 0.2M NaH 2 PO 4 (Merck, No. 6) 24 g / l until to obtain a pH 7.5 mg of

X.glucuronide (Clontech) are dissolved in 50μl of dimethylformamide, and 5ml of 0.2M NaPO4, pH7 (Solution A + B) is then added to obtain the reaction buffer.

2 / Realization of the test

200 μl of reaction buffer are deposited in the wells of an Elisa plate (96 wells), or 700 μl for a 24-well plate. Tissue fragments (stems, leaves, etc.) cut very thinly with a scalpel are then added. The plate is incubated in an oven at 37 ° C for about 18 hours. The appearance of a blue color reveals the activity of the enzyme GUS. Several rinses with 95 ° alcohol can be performed to discolor the tissue fragments, to facilitate reading

(optional).

The intensity of blue coloration is recorded according to the following scale:

0 null

1 +

2 ++

3 +++

Protocol for the preparation of tissue sections for plants transformed with the pAgMT2-GUS-tNOS construct

Positive GUS stems are vertically included in a 6% agar pad; the pad is then glued to the vibratom plate (an electronic device equipped with a razor blade to make thin sections of 1 μm), to obtain 40 μm sections in our study.

These sections are recovered on a slide in a drop of a 50/50 water / glycerol mixture; they are then observed between lamella and lamella under an optical microscope (LEICA type).

Results

Tomato plants (Solanum lycopersicum) of the KEMER genotype were transformed according to the protocol described above with Agrobacterium EHA 105 containing the plasmid pBI101 with the T-DNA comprising the construct pAgMat3-Gus-tnos or pAgMT2-Gus-tNos. Ten to twenty independent transformation events were obtained for each construct (To plants).

PCR assays were performed on TO plants using primers that amplify an overlapping sequence between the promoter of interest and the GUS gene, to control whether the plants have actually been transformed and include the construct.

The To PCR positive plants were repotted, and their seeds were harvested (T1 seeds) and sown on a growth medium supplemented with kanamycin (100 mg / L) to produce the plants.

T1. PCR analysis and segregation tests and KHI2 were performed on tomato plants to keep only T1 plants with 3: 1 segregation (KHI2 less than 5, indicating mono insertion). Six T1 plants per event were repotted for observation of the expression of the promoters studied (GUS tests).

I / Saline stress and GUS test on plants transformed with the pAgMat3-GUS-tNOS construct

^ Transformation with the construct pAqMat3-GUS-tNOS

PCR analyzes were performed using the following primers:

Promoter primer:

5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTGAACAGAAACAATTGTGGATG-S '(SEQ ID 36) GUS Primer:

5'-CGATCCAGACTGAATGCCC-3 ^J (SEQ ID 37)

Transformation events for which the PCR test is positive and T1 plants with 3: 1 segregation (KHI2 <5) are shown in Table 4.

Table IV: Transformation Events with the pAgMat3-GUS-tNOS PCR Construct Positive and Showing a Khi2 <5 Test

2) Salt stress and GUS test

The T1 plants obtained are sprayed with water at 50 mM NaCl 2 times daily, 10 ml with each watering, for 4 days. On the fifth day, the tissue samples are taken and the GUS tests are performed. Two checks are also carried out; a GUS positive control under the control of a constitutive promoter, a control of non-transformed KEMER plants. Eleven independent transformation events (Khi2 test less than 5, indicating a mono insertion) and 2 controls (non-transformed and Gus positive) were analyzed. When sufficient plants are available, 6 T1 plants per transformation event were repotted and used for the Gus: 4 salt stress (S) and 2 watered (NS) tests. If there is less

6 plants, all available plants were used.

The Gus test was performed separately on root and leaf fragments. The reading took place 3 days after the test (see plates in Figure 14).

Transformed plants showed a blue color in the roots. The preferential expression of GUS in the root tissue indicates activity of the AgMatS promoter in this tissue.

The results of this test are shown in Table 5 below.

Table V; GUS test on plants transformed with the built pAgMat3-GUS-tNOS and subjected or not to salt stress.

All of these data indicate a specific root expression of the AgMat3 promoter.

There / Test GUS on plants transformed with the built pAgMT2-GUS-tNOS

1) Transformation with the pAgMT2-GUS-tNOS construct The PCR analysis was carried out using the following primers: Promoter primer:

5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTGACCCACTATCAACAATGATC-S '(SEQ ID 38) GUS primer: δ'-CGATCCAGACTGAATGCCC-S' (SEQ ID 37)

Transformation events for which the PCR test is positive and T1 plants with 3: 1 segregation (KHI2 less than 5) are shown in Table 6.

Table VI: Transformation Events with the pAgMT2-GUS-tNOS PCR Construct Positive and Showing a Khi2 <5 Test

2) GUS test

Eleven independent transformation events (Khi2 test less than 5, indicating a mono insertion) and 2 controls (non-transformed and Gus positive) were analyzed. Six T1 plants per transformation event were repotted and used for Gus tests.

The Gus test was performed on fragments of stems and leaves (example of plate: Figure 15).

The results of this test are shown in Table 7 below.

Table VII; GUS test on plants transformed with the pAgMat3-GUS-tNOS construct and subjected or not to salt stress

Positive Gus fragments were then used for histo-cytological analysis and observation of vascular tissue staining. The reading took place 4 days after the test. The first observations show a blue color rather concentrated on the vascular tissues of the stem, in some cases, only on the veins of the leaves (figure 16).

3) Tissue cuts

Figure 17 shows tissue sections of plants 13 (a) and 103 at leaf (b) and stem (c). These cytological data indicate an expression at the level of phloem tissues. All of these data indicate a specific phloem expression of the AgMT2 promoter. The SEQ ID sequences identified in the present application are as follows:

SEQ ID No. 1: promoter sequence derived from the AgMaT3 gene of Apium graveolens;

SEQ ID No. 2: promoter sequence derived from the AgMT2 gene of Apium graveolens;

SEQ ID No. 3: promoter sequence derived from AgMT3 gene of Apium graveolens;

SEQ ID NO: 4: Complete 5 'sequence from AgMaT3 gene of Apium graveolens;

SEQ ID No. 5: complete 5 'sequence from AgMT2 gene of Apium graveolens;

SEQ ID N ^C 6: complete 5 'sequence from the AgMT3 gene of Apium graveolens;

SEQ ID No. 7: complete sequence of the AgMaT3 gene;

SEQ ID NO: 8 putative protein AgMaT3 deduced from the corresponding DNA sequence;

SEQ ID NO: 9: complete sequence of the AgMT2 gene;

SEQ ID NO: 10 putative protein AgMT2 deduced from the corresponding DNA sequence;

SEQ ID NO: 11: complete sequence of the AgMT3 gene;

SEQ ID NO: 12 putative protein AgMT3 deduced from the corresponding DNA sequence;

SEQ ID NO: 13: sequence of the AgMaT3 gene, without the promoter part;

SEQ ID NO: 14: cDNA encoding AgMaT3 protein (SEQ ID NO: 8).

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Claims

claims A nucleic acid sequence comprising all or part of the sequence SEQ ID No. 1, 2 or 3, such that: said nucleic acid sequence has a transcriptional promoter activity, and said part comprises at least 30 consecutive nucleotides of SEQ ID No. 1, 2 or 3. The sequence of claim 1, wherein said portion comprises at least 50 consecutive nucleotides of SEQ ID NO: 1, 2 or 3. The sequence of claim 1 or 2 having transcriptional promoter activity in plant cells. The sequence of claim 3, wherein said transcriptional promoter activity is induced by biotic or abiotic stress. The sequence of claim 3 or 4, wherein said sequence has transcriptional promoter activity preferentially in vascular tissues, especially in phloem and / or xylem. The sequence of claim 3 wherein said sequence has transcriptional promoter activity preferentially in the roots. 7. Sequence according to one of claims 3 to 6, having a transcriptional promoter activity in cells of celery (Apium graveolens L), Arabidopsis thaliana or tomato (Solanum lycopersicum L). 8. Sequence according to one of claims 1 to 7 comprising or consisting of a sequence selected from the sequences SEQ ID No. 1, 2, 3, 4, 5 or 6. 9. A nucleic acid sequence having, with respect to a sequence according to claim 1, less than 5 point mutations. 10. Sequence of nucleic acids having a transcriptional promoter activity and having at least 70% identity with one of the sequences SEQ ID No. 1, 2 and 3, preferably at least 80% identity, preferably 90% identity. The nucleic acid sequence according to one of claims 1 to 10, wherein said sequence is double-stranded DNA or single-stranded DNA. 12. Sequence of nucleic acids complementary to a sequence according to one of claims 1 to 11. 13. Nucleic acid sequence according to one of claims 1 to 12 for use in transgenic plants. 14. Nucleic acid probe capable of hybridizing with SEQ ID No. 1, 2 or 3 under conditions of high stringency, said probe having at least 25 nucleotides. A DNA construct comprising or comprising a promoter sequence according to any one of claims 1 to 11, and a sequence of interest to be transcribed downstream, such that the transcription of said sequence to be transcribed is under the control of the promoter sequence. The construct of claim 15, wherein said promoter sequence is heterologous with respect to the sequence of interest to be transcribed. The construct of claim 15 or 16, wherein said sequence of interest is a coding sequence. 18. A construct according to one of claims 15 to 17, wherein said construct is a vector. 19. Constructed according to one of claims 15 to 18, wherein said construct is a plasmid. 20. Plant cell transformed by a construct according to one of claims 15 to 19. The cell of claim 20 wherein said sequence is integrated into the genome of the cell. 22. Transgenic plant comprising in its genome a sequence according to claim 1 exogenous with respect to the plant. The plant of claim 22 wherein said sequence is inserted into the nuclear genome in stable form. The plant of claim 22 wherein said sequence is inserted into the mitochondrial or chloroplast genome. 25. Transgenic plant comprising cells according to claim 20 or 21, or part of such a plant, including seeds, reproductive material, seeds, roots, flowers or fruits, said parts being transgenic. The plant of claim 22, wherein said plant is a monocotyledonous plant. The plant of claim 22, wherein said plant is a dicotyledonous plant. 28. The plant according to claim 22, wherein said plant is a plant of agronomic interest and in particular a plant of the Cucurbitaceae, Chenopodiaceae, Cruciferae, Poaceae, Leguminosae, Apiaceae, Rosaceae, Valerianaceae, Solanaceae or Asteraceae family. Plant according to claim 23, wherein said plant is a tomato plant, a melon plant or a lettuce. 30. A process for preparing a transgenic plant comprising: a. Obtaining a construct according to one of claims 15 to 19; b. Introducing the construct into a cell from a plant of interest, c. Regeneration of a transgenic plant, d. Optionally, the proliferation of the plant to obtain an offspring. 31. Plant according to claim 22, obtained at the end of step d) of the process according to claim 30. 32. Use of a sequence according to any one of claims 1 to 11, or a construct according to one of claims 15 to 19 for the production of transgenic plants. A transgenic plant comprising in its genome a nucleic acid sequence comprising all or part of SEQ ID No. 1, such as said sequence has transcriptional promoter activity; said portion comprises at least 30 consecutive nucleotides of SEQ ID NO: 1, and said sequence is in functional association with a heterologous coding sequence and expressing said coding sequence specifically in the roots. 34. Transgenic plant according to claim 33, wherein said plant belongs to the family Apiaceae, Asteraceae, Brassicaceae, Chenopodiaceae, Cucurbitaceae, Poaceae, Rosaceae, Solanaceae, Valerianaceae or Leguminosae. A transgenic plant comprising in its genome a nucleic acid sequence comprising all or part of SEQ ID No. 1, 2 or 3 such that said sequence has transcriptional promoter activity, said portion comprises at least 30 consecutive nucleotides of SEQ ID NO: 1, 2 or 3, and said sequence is in functional association with a heterologous coding sequence and expressing said coding sequence specifically in the phloem. 36. Transgenic plant according to claim 35, wherein said sequence is SEQ ID 2 or part of SEQ ID 2 comprising at least 30 consecutive nucleotides, and wherein said plant belongs to the family Apiaceae, Asteraceae, Brassicaceae, Chenopodiaceae, Cucurbitaceae , Poaceae, Rosaceae, Solanaceae, Valerianaceae or Leguminosae. 37. The transgenic plant according to claim 35, wherein said sequence is SEQ ID 1 or 3 or a part of SEQ ID 1 or 3 comprising at least 30 consecutive nucleotides, where said plant belongs to the family Brassicaceae, and where the expression of said coding sequence is induced by biotic or abiotic stress. 38. The transgenic plant according to claim 37, wherein said biotic or abiotic stress is salt stress.