FR2840321A1 - SPECIFIC PROMOTER OF PLANT TISSUES FORMING SECONDARY WALLS - Google Patents

Abstract

The invention relates to a promoter specific for plant tissues involved in the formation of secondary walls, and comprising the sequences for regulating the transcription of the promoter of the AtBXL1 gene of Arabidopsis thaliana, and to its uses for expressing genes of interest in plants.

Description

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 The invention relates to a specific promoter for plant tissues which sets up secondary walls.

 The cell walls of plants ensure the maintenance of the structural integrity of plant cells and the mechanical resistance of plant tissues; they also play an important role in intercellular signaling.

 Cell walls are dynamic structures whose composition and morphology change during the development and differentiation of cells.

 In plants, there are two types of cell walls: the primary, elastic walls, which are synthesized during cell growth at the start of plant development, and the secondary, rigid and thick walls, which are put in place at the end. of growth and differentiation.

 The walls of the two types are mainly composed of polysaccharides (cellulose, hemicelluloses and pectins), and aromatic compounds, in particular lignins; they also contain proteins.

However, the composition, the proportions, and the structural organization of these constituents are different in the primary walls and the secondary walls.

 The composition and structure of secondary walls have a major influence on the qualities of plants in the field (for example resistance to lodging or attack by phytopathogens) as well as in the context of various industrial uses. For example, the content of lignin and hemicellulose, as well as the organization of these constituents within the secondary wall, play an essential role in the nutritional qualities of plants intended for human or animal consumption, as well as in the

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 properties of wood, or those of products based on cellulose fibers (textile, paper).

 For this reason, the modification by genetic engineering of the biosynthetic pathways of the constituents of the secondary wall is the subject of much research.

 It has thus been proposed to act on enzymes of the lignin biosynthesis pathway, in various applications such as improving the yield of paper pulp, or the digestibility of fodder [Cinnamoyl CoA reductase (CCR: PCT request WO 9712982; PCT application W09839454); caffeic acid 0-methyltransferase (COMT: PCT application WO9423044; OBA and ALLEN, J. Dairy Sci., 82, 135-142, 1999); Caféoyl Coenzyme A 3-0 methyltransferase (CCoAOMT: Application EP 0516958; et al., Transgenic Res. 10,457-464, 2001); CAD (LAPIERRE et al., Plant Physiol., 119,153- 164,1999); 4CL (HU et al., Nat. Biotech. 17,808-812, 1999).

 In the same type of applications, it has also been proposed to act on enzymes of the hemicellulose biosynthesis pathway, for example glucan synthetase (PCT application WO 99/67404) or UDP-Glucose-6 dehydrogenase ( PCT application WO 99/29875).

 Regulation of genes involved in the formation of walls has also been proposed to prevent lodging of cereals (for example, Fukei 71 gene, NISHIKUBO et al., Plant Cell Physiol., 41,776- 784, 2000).

 Conventionally, to act on an endogenous protein, an exogenous DNA sequence is expressed in the same cell, the transcription or translation product of which acts positively or negatively, depending on the application envisaged, on the expression or the activity of said protein. Examples include antisense suppression techniques or co-

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 suppression, which are currently frequently used to modulate the expression of various target genes.

 It is preferable, in a large number of cases, to target the expression of this exogenous sequence to certain cells and certain tissues, in order to avoid any interference with other functions of the plant.

Thus, to act on biosynthetic pathways of the constituents of the secondary walls, it is desirable to have promoters specific for these tissues.

 In the context of work concerning the genes involved in the biosynthesis of plant walls, the inventors have found that a protein, previously referenced on GenBank under the accession number BAB09906 as part of the complete sequencing of the genome of Arabidopsis thaliana, was a P-xylosidase specifically expressed in the parietal fraction. The gene encoding this protein is hereinafter called AtBXL1.

 The inventors also isolated the promoter from the AtBXL1 gene, and expressed the reporter gene uidA (GUS) coding for P-glucuronidase under the control of this promoter. They thus observed that the expression of the reporter gene was restricted to tissues (xylem and lignified fibers) containing secondary walls during formation.

 The 1988 bp sequence upstream of the translation initiation codon of the AtBXL1 gene, and which includes the promoter of said gene, is represented in the sequence list in the appendix under the number SEQ ID NO: 1.

 A promoter with an expression profile similar to that of the AtBXL1 promoter has been described previously. it is the promoter of the EgCAD gene (FEUILLET et al., Plant Molecular Biology, 27,651-654, 1995; SAMAJ et al., Planta, 204 (4), 437-443,1998).

However, analysis of the sequences of these promoters shows that they have a different structure: the

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 promoter of the AtBXL1 gene has an ethylene response box (ERE, located at-164 upstream of the translation initiation codon) and a gibberellin response box (P-Box, en-289 upstream from the codon translation initiation) but no auxin response box; the promoter of the EgCAD gene has response boxes to auxin (AuxRR-Core, en-305 upstream of the translation initiation codon), and to abscissic acid (ABRE, in-157 upstream of the codon translation initiation) but no ethylene response boxes, or gibberellin response boxes.

 The subject of the present invention is a polynucleotide constituting a promoter specific for the plant tissues involved in the formation of secondary walls, characterized in that it comprises the sequences for regulating the transcription of the promoter of the AtBXLl gene from Arabidopsis thaliana.

 According to a preferred embodiment of a promoter in accordance with the invention, it comprises the polynucleotide of sequence SEQ ID NO: 1.

 The present invention encompasses any promoter consisting of a nucleic acid fragment comprising the functional domains of the promoter of the AtBXL1 gene.

 Those skilled in the art can, from the polynucleotide of sequence SEQ ID NO: 1, identify these functional domains, as well as the DNA motifs involved in the function of each of them, by techniques known in themselves. promoter dissection, for example by determining the level and specificity of the promoter activity of deletion mutants of this polynucleotide, and / or by techniques such as those of DNA fingerprints, by incubating the polynucleotide of sequence SEQ ID NO: 1, or portions thereof, with nuclear extracts from tissue cells involved in the formation of

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 secondary walls, as well as with nuclear extracts from cells of tissues in which the promoter of the AtBXL1 gene is inactive.

 The invention also includes: - the expression cassettes, comprising, in addition to a promoter according to the invention, a gene of interest placed under transcriptional control of said promoter, or a site allowing the insertion of said gene of interest; - the recombinant vectors, resulting from the insertion of a promoter or of an expression cassette in accordance with the invention in a host vector.

 A promoter according to the invention can be used to specifically express a polynucleotide of interest in the plant tissues involved in the formation of secondary walls.

 Said polynucleotide of interest can in particular be a DNA sequence making it possible to modulate the expression of an enzyme of the biosynthesis pathway of the constituents of the secondary wall, for example by antisense co-suppression or suppression, or by production of interfering RNA .

 It may also be any gene coding for a protein of interest which it is desired to express specifically at the level of the secondary walls.

 The invention further relates to plant cells and transgenic plants transformed with at least one nucleic acid molecule comprising a promoter according to the invention.

 The transformed cells and the transgenic plants in accordance with the invention can also be used as models for studying and / or modifying the expression of different genes at the level of the secondary walls.

 The present invention will be better understood with the aid of the additional description which follows, which is

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 refers to nonlimiting examples illustrating the obtaining of the promoter of the AtBXL1 gene, and its use for expressing a heterologous gene in the tissues involved in the formation of secondary walls.

EXAMPLE 1 INSERTION OF HETEROLOGOUS GENES UNDER CONTROL OF THE ATBXLI GENE PROMOTER
The promoter of the AtBXL1 gene is amplified by PCR from genomic DNA of Arabidopsis thaliana, Wassilevskija ecotype (WS), using the following primers: P1: 5'-GGG GCT GCA GGT GCT TTA TTA TAA CAA GAC AT-3 'P2: 5'-CCC CCA AGC TTA ATT CGT TCT AGT GTT TTC-3'
Amplification is carried out using Pfu polymerase (PROMEGA) according to the protocol recommended by the supplier. The sequence of the amplification product is represented in the sequence list in the appendix under the number SEQ ID NO: 1.

 This amplification product is cloned into the plasmid pBSKS- (STRATAGENE).

1) Insertion of the uidA gene under the control of the promoter of the AtBXL1 gene:
The plasmid pBSKS- containing the promoter of the AtBXL1 gene is digested with PstI and HindIII and the PstI-HindIII fragment containing the promoter of the AtBXL1 gene is cloned between the corresponding sites of the plasmid pCAMBIA1391Xb, in phase with the coding sequence uidA (GUS) coding for P-glucuronidase (JEFFERSON, Ann. NY Acad. Sci., 700.53-73, 1993).

pCAMBIA1391Xb(promAtBXLI), est schématisée sur la Figure 1. The resulting construction, hereinafter referred to as

pCAMBIA1391Xb (promAtBXLI), is shown diagrammatically in Figure 1.

Figure 1 legend: P35S: CaMV 35S promoter; T35S: CaMV terminator sequence; hpt: sequence

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 encoding hygromycin phosphotransferase; AtBXL1 promoter: AtBXL1 promoter sequence; uidA: coding sequence for ss-glucuronidase; TNos: terminator of nopaline synthase; BG: left border of T-DNA; BD: right border of T-DNA.

2) Insertion of the cDNA of the CCR of Arabidopsis thaliana in antisense orientation under the control of the promoter of the AtBXL1 gene;
The cDNA of the AtCCRl gene (GenBank access no .: AF320624) is inserted, in antisense orientation, in the plasmid pCAMBIA1391Xb (promAtBXL1) in replacement of the coding sequence uidA.

 The construction obtained is shown diagrammatically in Figure 2.

Figure 2 legend: P35S: CaMV 35S promoter; T35S: CaMV terminator sequence; hpt: sequence coding for hygromycin phosphotransferase; AtBXL1 promoter: AtBXL1 promoter sequence; AtCCRl: AtCCRI cDNA in antisense orientation; TNos: terminator of nopaline synthase; BG: left border of T-DNA; BD: right border of T-DNA.

3) Insertion of the poplar F5H cDNA in sense orientation under the control of the promoter of the AtBXL1 gene:
The poplar F5H cDNA (GenBank access no .: AJ010324) is inserted, in sense orientation, in the plasmid pCAMBIA1391Xb (promAtBXL1) in replacement of the coding sequence uidA.

 The construction obtained is shown diagrammatically in Figure 3.

Figure 3 legend: P35S: CaMV 35S promoter; T35S: CaMV terminator sequence; hpt: sequence coding for hygromycin phosphotransferase; AtBXL1 promoter: AtBXL1 promoter sequence; F5H: cDNA of poplar F5H in sense orientation; TNos: terminator

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 nopaline synthase; BG: left border of the DNAD; BD: right border of T-DNA.

 These different constructions are transferred into bacteria of the C58pMP90 strain of Agrobacterium tumefaciens (KONCZ and SCHELL, Mol Gen Genet 204: 383-396, 1986) for the transformation of plants.

EXAMPLE 2 OBTAINING TRANSGENIC PLANTS EXPRESSING A HETEROLOGOUS GENE UNDER CONTROL OF THE ATBXL1 GENE PROMOTER.

 The transformation of Arabidopsis thaliana plants is carried out by soaking the flower buds in the solution of Agrobacterium tumefaciens, as described by CLOUGH and BENT, (Plant J., 16,735-743, 1998).

 The primary transformants are selected on Arabidopsis medium (ESTELLE and SOMMERVILLE, Mol. Gen.

Broom. 206,200-206, 1987) containing 50 mg / L of hygromycin, and self-pollinated in order to obtain individuals homozygous for insertion. The seeds from self-pollination are harvested after the stems are completely dried. Only lines segregating 3: 1 for resistance to hygromycin and having a single homozygous insertion are kept for the analysis of the expression of the heterologous gene by detection of its activity.

1) Expression of the uidA gene
The activity (3-glucuronidase is sought by histochemical analysis on sections of different organs (roots, leaves, flowers and pods) of freshly harvested plants. The detection is carried out as described by JEFFERSON et al. (EMBO J. 6,3901 -3907, 1987), in the presence of 5-bromo-4-chloro-3-indolyl p- D-glucuronic acid (XGluc, BIOSYNTH AG), in 100mM potassium phosphate buffer, pH7, containing 0.1% of Triton X100, as well as 10MM of K3Fe (CN) 6 and 10MM of K4Fe (CN) 6 to avoid the diffusion of the intermediate products of the reaction.

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 A specific expression of ssglucuronidase is observed in the vascular tissues and fibers of all the organs tested, and in particular in the xylem (protoxylem and metaxylem) and the intrafascicular cambium. No expression is observed in the other tissues.

 This distribution corresponds to a specific expression of the tissues in which the formation of secondary walls takes place.

2) Expression of the AtCCR cDNA in antisense:
The decrease in CCR activity resulting from the expression of the cDNA of AtCCR1 in antisense is sought in homozygous transformants by a biochemical test, as described by CHABANNES et al. (Plant J., 28,257-270, 2001 ), and evaluated by the quantitative and qualitative analysis of lignins from mature flower stalks.

 The quantitative analysis is carried out by Klason test (DENCE et al., Lignin determination, p 33-61, Methods of Lignin Chemistry, C. DENCE, S. LIN eds, SPRINGER VERLAG, Berlin, 1992).

 The qualitative analysis is carried out by thioacidolysis (LAPIERRE et al., Res. Chem. Intermed. 21, 397,412, 1995).

 A decrease in CCR activity results in a decrease in the amount of total lignins, an increase in the S / G ratio, and a decrease in beta-0-4 labile bonds as described by PIQUEMAL et al. (Plant J. , 13,71-83, 1998).

3) Expression of F5H:
The F5H activity resulting from the expression of poplar F5H is sought in homozygous transformants by a biochemical test (reaction of MAULE) on thin sections of mature flower stalks, and by quantitative and qualitative analysis of the lignins of the mature stems of homozygous transformants.

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 The quantitative and qualitative analyzes of lignins are carried out as indicated above for the CCR.

 The F5H activity results in the presence of S units of lignin in the xylem vessels, which is manifested by a red coloration of the xylem by the reaction of MULE, by the increase in the S / G ratio of the total lignins, and the increase in beta-0-4 labile bonds as described by FRANKE et al. , Plant J., 22 (3), 223-234, 2000).

Claims (7)

1) Polynucleotide constituting a specific promoter of the plant tissues involved in the formation of secondary walls, characterized in that it comprises the sequences for regulating the transcription of the promoter of the AtBXLl gene from Arabidopsis thaliana.  2) Polynucleotide according to claim 1 characterized in that it comprises the sequence SEQ ID NO: 1.  3) Expression cassette characterized in that it comprises a polynucleotide according to any one of claims 1 or 2.  4) Recombinant vector characterized in that it comprises at least one polynucleotide according to any one of claims 1 or 2.  5) Plant cell transformed with at least one polynucleotide according to any one of claims 1 or 2.  6) Transgenic plant transformed with at least one polynucleotide according to any one of claims 1 or 2.  7) Use of a polynucleotide according to any one of claims 1 or 2 to express a polynucleotide of interest in a plant tissue involved in the formation of secondary walls.