EP2440665A1 - Production of plants with reduced lignin content - Google Patents

Abstract

The invention relates to the production of plants having a reduced lignin content and in which the cellulose hydrolysis of the walls is increased, via the total or partial inhibition of the expression and/or the activity of two laccases in said plant.

Description

 OBTAINING PLANTS WITH REDUCED LIGNIN CONTENT

The present invention relates to a method for selecting or obtaining plants having a reduced lignin content.

Lignins are insoluble polymers located in the plant walls and resulting from the polymerization of 3 phenolic monomers (or monolignols), derived from the phenylpropanoids route (NEISH, Constitution and Biosynthesis of Lignin, New York editions: Springer Verlag, 1-43 , 1968). Their biosynthetic pathway is complex and involves different steps, part of which is carried out in the cytoplasm (synthesis of monolignols) and another in the wall (polymerization). The p-coumaryl, coniferyl and sinapyl alcohols are the respective precursors of the p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) units constituting the lignins. These precursors are oxidized to phenolic radicals which spontaneously couple by various bonds, which leads to the formation of lignins. Among the interunit links, there are labile bonds, called β-O-4 and resistant bonds. During the polymerization, other bonds can also be established with other parietal compounds (polysaccharides and proteins) to form a complex three-dimensional network. The formation of phenolic radicals would be catalyzed by oxidases, such as peroxidases, laccases or other oxidases. A large number of these enzymes in combination with regulatory proteins would be required for the assembly of H, G and S units (BOUDET, Plant Physiol Biochem, 38, 81-96, 2000, see for review: RALPH et al. ., Lignins, Encyclopedia of Life Sciences, John Wiley & Sons, 2007). However, these enzymes are still poorly identified because they belong to multigene families (BARRIERE et al., Genes, Genomes and Genomics, Global Siences Books, 2007, Review). Although the mechanisms involved in vivo in the biosynthesis of lignins are not fully understood, it is generally considered that laccases could be involved in the first stages of polymerization, for the formation of dimers or trimers, while the peroxidases would make it possible to obtain a higher degree of polymerization from dimers and trimers (ROS BARCELO, International Review of Cytology, 176, 87-132, 1997). The lignin content of plants has a major influence on their industrial uses. For example, it affects the nutritional value of plants for animal feed, as well as the performance of paper processes (yield and quality of the pulp obtained) and the production yield of biofuel. Indeed: An important component of the nutritional value of forage plants, such as forage corn, is digestibility. Thus, cows fed on more digestible varieties show an increase in milk production and better weight gain. In addition, these more digestible varieties allow animals to reach their potential with a lower level of complementation, which in particular makes it possible to reduce production costs. An important factor limiting the digestibility of forage plants is related to the presence in the walls of plant cells of lignins: lignins establish different types of linkages with other parietal constituents (including polysaccharides) and hinder the accessibility of digestive enzymes polysaccharides (carbohydrates), the main sources of energy for herbivores;

- most of the paper pulp is obtained by chemical delignification of the wood, in order to isolate the vegetable fibers consisting mainly of cellulose and bound in them by the lignins. However, this step of delignification is very demanding in energy and reagents

(acids in particular), because of the resistance of lignins to chemical degradation. In the case of thermomechanical pulp production, lignins that are not removed are responsible for the photojaunissement of the paper. Lignins rich in units

S are more sensitive to delignification papermaking processes since the S units are mostly linked by β-O-4 bonds (target bonds of these processes). As a result, in the case of hardwoods used by paper mills, a higher content of β-0-4 bound S units is sought in the paper industry to improve the efficiency of paper cooking (GUERRA et al., Ind. Eng Chem Res., 47, 8542-8549, 2008);

- biofuels are formed from bioethanol (a product miscible with gasoline) or oil

(to make a diesel product). The production of bioethanol, currently produced from starch or sucrose, could also be made from the cellulose of wood or straw. In this case, the production process comprises the following steps: acid pretreatment of the raw material to break the interactions between lignins and polysaccharides (this pretreatment facilitates the action of hydrolytic enzymes which transform the polysaccharides of the wall into simple sugars), followed by a fermentation step of simple sugars, leading to the production of bioethanol. However, the initial acid pretreatment results in the production of compounds capable of inhibiting the fermentation step. It has therefore been suggested that by modifying the lignins, the parietal polysaccharides would be more accessible to the hydrolytic enzymes. This would not only eliminate or limit acid pretreatment and the associated problems of fermentation inhibition, but also reduce the environmental impacts associated with the residues of the acid treatments. In this context, the quantitative modification of lignins in plants is the subject of much research. The qualitative modification of the lignins (modification of their structure or their interactions with the other polymers of the walls) is also very studied. For example, lignins rich in S units and β-O-4 bonds are much easier to remove in the production of pulp by chemical means.

One of the preferred ways to reduce the lignin content in plants is the production of plants by genetic engineering. It has thus been proposed to act on the enzymes of the lignin biosynthetic pathway, such as laccases (International Application WO 97/45549), peroxidases (International Application WO 2004/080202) Cinnamoyl CoA reductase (CCR; WO 97/12982 and WO 98/39454), caffeic acid O-methyltransferase (COMT International Application WO 94/23044, OBA and ALLEN, J. Dairy Sci., 82, 135-142, 1999), Caféoyl Coenzyme A 3-0 methyltransferase (CCoAOMT, Application EP 0516958, GUO et al., Transgenic Res 10, 457-464, 2001), Cinnamyl alcohol dehydrogenase (CAD, LAPIERRE et al., Plant Physiol., 119, 153- 164, 1999), and 4-coumarate: coenzyme A ligase (4CL, HU et al., Nat Biotech 17, 808-812, 1999). With particular reference to laccases, International Application WO 97/45549 describes a tobacco laccase (the sequence of which is furthermore described by KIEFER-MAYER et al., Gene, 178, 205-207, 1996), and proposes to increase or reduce the amount of lignin produced by a plant by overexpressing said laccase (or a protein having at least 50% amino acids homologous to those of said laccase), or by inhibiting its expression.

In Arabidopsis thaliana, the multigene family of laccases has 17 members, 7 of which are expressed in the stems, the most lignified organ. The most highly expressed genes are LAC4 (At2g38080), LAC11 (At5g60020) and LAC2 (At2g29130). The LAC2 and LAC11 genes belong to the same subclass and the LAC4 gene belongs to a close subclass according to the phylogenetic trees published by POURCEL et al. (Plant Cell, 17, 2966-2980, 2005) and CAPARROS-RUIZ et al. (Plant Science, 171, 217-225, 2006). The LAC11 gene codes for the LAC17 protein (AtLAC17), the sequence of which is available under the accession number NM_125395 in the GENBANK database, and is also reproduced in the attached sequence listing under the identifier SEQ ID NO: 2. The LAC4 gene codes for the LAC4 protein (AtLAC4), the sequence of which is available under the accession number NM_129364 in the GENBANK database, and is also reproduced in the attached sequence listing under the identifier SEQ ID NO: 4. The LAC2 gene encodes the LAC2 protein (AtLAC2), the sequence of which is available under accession number NM_128470 (GI: 186503951) in the GENBANK database. In Arabidopsis AtLAC17 is expressed at interfascicular fibers. The inventors have thus demonstrated that the orthologous proteins of the AtLAC17 protein have at least 60% identity or at least 75% similarity with it and comprise, from the N-terminus to the C-terminus. terminal, at least one of the four sequence consensus peptide domains:

• HWHGI / VRQL (SEQ ID NO: 12, amino acids corresponding to positions 80-87 of the peptide sequence of AtLAC17) or HWHGI / VR / LQL / M / V (SEQ ID NO: 38), • I / VNAALNDELFF ( SEQ ID NO: 13, amino acids corresponding to positions 223-233 of the peptide sequence of AtLAC17) or INA / SALN / ED / N / EELFF (SEQ ID NO: 39),

• ESHPLHLHGF / YN / DFFVVGQGF / YGNF / YD (SEQ ID NO: 14, amino acids corresponding to positions 476-498 of the peptide sequence of AtLAC17), or ESHPL / FHL / MHGF / YN / DF / YF / YVV / IGQ / T / E-GF / V / TGNF / YD / N (SEQ ID NO: 40) and

• A-D-N-P-G-V-W (SEQ ID NO: 15, amino acids corresponding to positions 539-546 of the peptide sequence of AtLAC17), or A / V-D-N-P-G-V / O-W / O (SEQ ID No.

NO: 41), where "0" indicates that an amino acid is absent, respectively.

As non-limiting examples of orthologs of the L LAC17 protein. thaliana, we will mention in particular the laccases of:

-means (Zea mays) of SEQ ID NO: 5 and

6 (sequences also available under accession numbers NM_001112405.1 and EU957078 in the GENBANK database) and sequences available in the GENBANK database under accession numbers

GI: 162461426 (SEQ ID NO: 42), GI: 226503958 (SEQ ID NO:

43), GI: 226494660 (SEQ ID NO: 44), GI: 212721074 (SEQ ID

NO: 45), GI.162463584 (SEQ ID NO: 46) and GI.162461268

(SEQ ID NO: 47), - sugar cane (Saccharum offieinarum) of sequence SEQ ID NO: 7,

sorghum (Sorghum bicoior) of sequences SEQ ID NO: 8, 9, 10 and 11, of Brachypodium, such as the sequences Bradilg66720 (SEQ ID NO: 48), Bradi2g54680 (SEQ ID NO: 49), Bradilg24910 (SEQ ID NO: 50), Bradilg24880 (SEQ ID NO: 51), Bradi2g54740 (SEQ ID NO: 52), Bradi2g23370 (SEQ ID NO: 53), Bradi2g23350 (SEQ ID NO: 54) and Bradi2g54690 (SEQ ID NO: 55), rice, such as the sequences available in the GENBANK database under the accession numbers GI: 113548170 (SEQ ID NO: 56), GI.113534304

(SEQ ID NO: 57), GI: 113579298 (SEQ ID NO: 58),

GI.113579297 (SEQ ID NO: 59), GI: 113534303 (SEQ ID NO: 60), GI.113579295 (SEQ ID NO: 61), GI.255673866 (SEQ ID NO.

NO: 62) (these sequences have the referenced domain under access number IPR017761 in the InterPro database), and

of poplar, such as the sequences PtLACl (gene POPTR_0001sl4010.1, SEQ ID NO: 63), PtLAC40 (POPTR_0001s41160.1, SEQ ID NO: 64), PtLAC41 (POPTR_0001s41170.1, SEQ ID NO: 65), PtLAC6 ( POPTR_0001s41170.1, SEQ ID NO: 66), PtLAC24 (POPTR_0011sl2010.1, SEQ ID NO: 67) and PtLAC25 (POPTR_0011sl2100.1, SEQ ID NO: 68). The table represented in FIG. 5 shows the presence (indicated in the table by the sign "X") of the consensus peptide domains as defined above in the orthologous sequences of the AtLAC17 protein in Zea mays, S. officinarum, Sorghum bicolor, Brachypodum, poplar and rice, as well as the respective percentages of identity and similarity to AtLAC17.

The LAC17 protein has 55.2% identity with the LAC4 protein, and 67.1% identity with the LAC2 protein, but the latter does not include the consensus peptide domain of sequence SEQ ID NO: 14, and 54.6. % identity with the tobacco laccase described in International Application WO 97/45549; the LAC4 protein has 54.0% identity with the LAC2 protein and 75.8 identity with the tobacco laccase described in the International Application WO 97/45549 (it appears that said tobacco laccase is the orthologue of the AtLAC4 protein), the percentages of identity being calculated over the entire length of the sequences using the needle program (NEEDLEMAN and WUNSCH, J. Mol Biol., 48, 443-453, 1970) using the default parameters. : "Matrix": EBLOSUM62, "Gap penalty": 10.0 and "Extend penalty": 0.5. In Arabidopsis, AtLAC4 is expressed at the level of the xylem vessels and interfascicular fibers. As nonlimiting examples of orthologs of the LAC4 protein of A. thaliana, we will mention in particular the laccases of: Brachypodium, such as the sequence

Bradilg74320 (SEQ ID NO: 69), which has 67% identity and 83% similarity with the sequence SEQ ID NO: 4,

- rice, such as the sequence available in the GENBANK database under the accession number

GI: 150383842 (QOIQUl; SEQ ID NO: 70), which has 69% identity and 83% similarity to the sequence SEQ ID

NO: 4, and poplar, the sequences PtLAC14 (POPTR_0006s09830.1; SEQ ID NO: 71, which has 75% identity and 87% similarity with the sequence SEQ ID NO: 4), PtLAC15 (POPTR_0006s09840.1; SEQ ID NO: 72, which has 76% identity and 87% similarity to sequence SEQ ID NO: 4), PtLAC32 (POPTR_0016sll950.1, SEQ ID NO: 73, which has 78% identity and 88% identity. similarity with the sequence SEQ ID NO: 4) and PtLAC33

(POPTR_0016sll960.1; SEQ ID NO: 74, which has 77% identity and 87% similarity to the SEQ ID sequence

NO: 4). Lines of A. thaliana of the SALK collection in the CoIO (Columbia) accession with T-DNA insertions at the LACI1 gene (line SALK_016748), LAC4 (line SALK_051892) and LAC2 (line SALK_025690) have been identified. The mutants Iac4 and Iac2 have in particular been described by BROWN et al. (Plant Cell, 17, 2281-2295, 2005). These mutants show strongly reduced or zero mutated gene expression but do not exhibit a particular phenotype in the greenhouse.

The inventors have investigated whether these mutations have an effect on the amount of lignins of the mutated plants, and their qualitative (structural) properties. They found that the Iac2 mutant did not differ significantly from the CoIO wild line, but that the Iac4 and lacII mutants contained lignin (determined on mature dry stems) reduced from 6 to 8. % and had an increased cellulolytic yield of 17% in the case of lac11 and 52% in that of Iac4, relative to the wild line CoIO (cellulolysis carried out without acid pretreatment).

In addition, the inventors obtained by crossing, from mutants Iac4 and lac11, double mutants Iac4 / lac17. They found that these double mutants had a very small amount of lignin (about 19% compared to the CoIO wild line), and a better cellulolytic yield compared to the CoIO wild line (+ 25% to + 42% ) and the simple lacll mutant (+ 6% to + 21%) approximately.

Accordingly, the subject of the present invention is a process for reducing the lignin content of a plant and increasing cellulolysis of the walls of said plant, characterized in that the expression and / or the activity in said plant: a) a laccase of which the polypeptide sequence has at least 60% identity, and in order of increasing preference at least 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% and 99% identity, or at least 75% similarity, and in order of increasing preference at least 78%, 79%, 80%, 83%, 85%, 90% %, 95%, 97%, 98% and 99% of similarity with the sequence SEQ ID NO: 2 (LAC17), and comprises, from its N-terminal end to its C-terminal end, at least one of the 4 , preferably at least 2 of 4, more preferably at least 3 of 4 and more preferably the 4 consensus peptide domains i) to iv) respectively, i) the sequence consensus peptide domain SEQ ID NO: 12 or 38, ii) the consensus peptide domain of sequence SEQ ID NO: 13 or 39, iii) the consensus peptide domain of sequence SEQ ID NO: 14 or 40, iv) the sequence consensus peptide domain

SEQ ID NO: 15 or 41, and b) a laccase whose polypeptide sequence has at least 65% identity, and in order of increasing preference at least 70%, 75%, 80%, 85%,

90%, 95%, 97%, 98% and 99% identity, with the sequence SEQ ID NO: 4 (LAC4).

The term "lignin content" means the Klason lignin content. This content can be measured by the assay of the acid-insoluble lignin fraction (LAS) present in the parietal residue (RP) of a plant, as described in Example 3 below.

The term "laccase" refers to a copper (EC 1.10.3.2) enzyme that catalyzes the oxidation of a phenolic substrate by using dioxygen as the final acceptor of electrons. Unless otherwise specified, the identity percentages shown here are set, as above, using the needle program using the default settings.

The present invention is applicable to dicotyledonous or monocotyledonous plants. As non-limiting examples, it can be applied to corn, wheat, barley, rye, triticale, oats, rice, sorghum, sugar cane, poplar and pine.

By way of nonlimiting examples of laccases, as defined in paragraph a) above, mention may be made, in maize (Zea mays), of the peptide sequences SEQ ID NO: 5, 6 and 42 to 47, in cane sugar (Saccharum officinarum), the sequence SEQ ID NO: 7, in sorghum (Sorghum bicolor), the peptide sequences SEQ ID NO: 8 to 11, in Brachypodium, the peptide sequences SEQ ID NO: 48 to 55), in rice, the peptide sequences SEQ ID NO: 56 to 62, and in poplar, the peptide sequences SEQ ID NO: 63 to 68.

By way of nonlimiting examples of laccases, whose polypeptide sequence has at least 60% identity or at least 75% similarity with the sequence SEQ ID NO: 2 and comprises, from its N-terminal end towards at its C-terminal end, the 4 consensus peptide domains of SEQ ID NO: 12, 13, 14 and 15, respectively, in maize, the peptide sequences SEQ ID NO: 5, 6, 42, 43, 44 and 45, in the sugar cane, the peptide sequence SEQ ID NO: 7, in the sorghum, the peptide sequences SEQ ID NO: 8 (partial sequence of the protein), 9, 10 and 11, in Brachypodium, the SEQ peptide sequences ID NO: 50 and 51, in rice, the peptide sequence SEQ ID NO: 58, and in poplar, the peptide sequences SEQ ID NO: 63, 67 and 68.

By way of nonlimiting examples of laccases, whose polypeptide sequence has at least 65% identity with the sequence SEQ ID NO: 4, mention may be made in Brachypodium, the peptide sequence SEQ ID NO: 69, in rice, the peptide sequence SEQ ID NO: 70 and in poplar, the peptide sequences SEQ ID NO: 71 to 74.

The total or partial inhibition of the expression and / or the activity of a laccase as defined above can be obtained in various ways, by methods known per se.

In a particularly advantageous manner, this inhibition can be obtained by intervening upstream of the production of said laccase, by mutagenesis of the gene coding for this protein, or by inhibition or modification of the transcription or translation of this laccase.

Mutagenesis of the gene coding for said laccase may occur at the level of the coding sequence or of the expression regulation sequences, in particular of the promoter. For example, it is possible to delete all or part of said gene and / or to insert an exogenous sequence. By way of example, for maize, mention may be made of insertional mutagenesis: a large number of individuals derived from an active plant are produced for the transposition of a transposable element (AC element or Mutator), and selected by example by PCR, the plants in which an insertion was carried out in the gene of said laccase. This exogenous sequence may also be a T-DNA (fragment of the Agrobacterium tumefaciens Ti plasmid).

It is also possible to introduce one or more point mutations with physical agents (for example radiation) or chemical agents. These mutations have the consequence of shifting the reading frame and / or of introducing a stop codon into the sequence and / or of modifying the level of transcription and / or translation of the gene and / or of making the enzyme less active than the wild-type protein. The mutated alleles of the gene of said laccase can be identified for example by PCR using primers specific for said gene.

In this context, one can notably use techniques of the type "TILLING" (Targeting Induced Local In Genomes lesions, McCALLUM et al, Plant Physiol., 123, 439-442, 2000).

It is also possible to perform site-directed mutagenesis targeting the gene encoding said laccase. The inhibition or the modification of the transcription and / or the translation can be obtained by the expression of sense, antisense or double-stranded RNA derived from the gene of said laccase, or the cDNA of this protein, or else by the use of interfering RNAs (for review on antisense inhibition techniques see for example: WATSON and GRIERSON, Transgenic Plants: Fundamentals and Applications (HIATT, A, ed) New York: Marcel DEKKER, 255-281, 1992 CHICAS and MACINO, EMBO reports, 21, 992-996, 2001, for a review relating more specifically to the use of interfering RNAs (HANNON, Nature, 418, 244-251, 2002).

The present invention also relates to a recombinant DNA construct comprising one or more polynucleotides capable of inhibiting the expression of the two laccases as defined above. By way of nonlimiting examples, said polynucleotides can encode antisense RNAs, interfering RNAs (non-coding double-stranded RNA with a length of approximately 21 to 25 nucleotides), microRNAs (non-coding single-stranded RNAs of a length of about 21 to 25 nucleotides) (OSSOWSKI et al., The Plant Journal, 53, 674-690, 2008; SCHWAB et al., Methods Mol Biol., 592, 71-88, 2010, WEI et al., Funct Integr Genomics, 9, 499-511, 2009) or ribozymes targeting the gene encoding a laccase as defined above.

Preferably, said polynucleotides capable of inhibiting the expression of LAC4 and LA17 laccases as defined above, are microRNAs, such as microRNAs miR397 and miR408, preferably miR397. As non-limiting examples of such micro-RNAs, those of the following sequences can be used:

- SEQ ID NO: 75 (AtmiR397a) or SEQ ID NO: 76 (AtmiR397b), obtained from Arabidopsis thalina (ABDEL-GHANY and PILON, J Biol Chem., 283, 15932-15945, 2008);

SEQ ID NO: 77 (ptc-miR397), obtained from poplar; or

SEQ ID NO: 78 (Bdi-miR397a) or SEQ ID NO: 79 (Bdi-miR397b), obtained from Brachypodium distachyon

(ZHANG et al., BMC Genomics, 23, 10: 449, 2009, UNVER and BUDAK, Planta, 230, 659-669, 2009).

According to a preferred embodiment of the invention, the recombinant DNA construct is chosen from:

a DNA construct comprising a fragment of at least 15 consecutive nucleotides, preferably at least 20 consecutive nucleotides of the cDNA of the gene encoding a laccase as defined above, a DNA construct of 200 to 1000 bp, comprising a fragment of the cDNA of the gene coding for a laccase as defined above, or a complementary polynucleotide, which when it is transcribed forms a hairpin RNA (hairpine RNA or ribozyme) targeting said gene ; a DNA construct, capable, when transcribed, of forming a microRNA targeting the gene coding for a laccase as defined above.

According to a particular embodiment of the invention, said recombinant DNA construct comprises a fragment of at least 15 consecutive nucleotides, preferably at least 20, and most preferably at least 50 consecutive nucleotides of a polynucleotide. of sequence SEQ ID NO: 1 or SEQ ID NO: 3, or of a polynucleotide complementary to a polynucleotide of sequence SEQ ID NO: 1 or SEQ ID NO: 3.

These constructions can be notably:

expression cassettes, comprising one or more recombinant DNA constructs as defined above, under transcriptional control of a suitable promoter. These expression cassettes may also advantageously comprise other regulatory elements, in particular transcription regulation elements such as terminators, amplifiers, and the like. ; recombinant vectors, comprising one or more recombinant DNA constructs as defined above, or advantageously an expression cassette as defined above.

Recombinant DNA constructs in accordance with the invention may also comprise other elements, for example one or more selection markers.

Those skilled in the art have a very wide choice of elements that can be used to obtain recombinant DNA constructs in accordance with the invention. As non-limiting examples of promoters usable in the context of the present invention, mention may be made of: constitutive promoters, such as the 35S promoter of cauliflower mosaic virus (CaMV) described by KAY et al. (Science, 236, 4805, 1987), or its derivatives, the promoter of the cassava vein mosaic virus (CsVMV) described in the International Application WO 97/48819, the corn ubiquitin promoter or the rice "Actin-Intron-actin" promoter (McELROY et al., Mol Gen. Genet., 231, 150-160, 1991; GenBank no. access S 44221). inducible or tissue-specific promoters, in order to modify the lignin content or composition only at certain stages of the development of the plant, under certain environmental conditions, or in certain target tissues, for example, the stems, leaves, seeds, husks, cortex or xylem (eg, the cinnamyl alcohol dehydrogenase (CAD) promoter or the 4 coumarate-CoA ligase (4CL)).

As non-limiting examples of other transcriptional regulation elements usable in the context of the present invention, mention will be made of terminators, such as the 3'NOS terminator of nopaline synthase (DEPICKER et al., J. Mol Appl., Genet., 1, 561-573, 1982), or the 3'CaMV terminator (FRANCK et al., CeIl, 21, 285-294, 1980, GenBank accession number V00141).

As non-limiting examples of selection marker genes that can be used in the context of the present invention, mention will be made in particular of genes conferring resistance to an antibiotic (HERRERA-ESTRELLA et al., EMBO J., 2, 987-995 , 1983) such as hygromycin, kanamycin, bleomycin or streptomycin, or a herbicide (EP 0 242 246) such as glufosinate, glyphosate or bromoxynil, or the NPTII gene which confers kanamyine resistance. (BEVAN et al., Nucleic Acid Research, 11, 369-385, 1984).

Plant transformation can be carried out by many methods, known in themselves to those skilled in the art.

For example, it is possible to transform plant cells, protoplasts or explants, and to regenerate an entire plant from the transformed material. The transformation can thus be carried out, by way of non-limiting examples: by transfer of the vectors according to the invention in protoplasts, in particular after incubation of the latter in a solution of polyethylene glycol (PEG) in the presence of divalent cations

(Ca ²⁺ ) according to the method described in the article by KRENS et al. (Nature, 296, 72-74, 1982); by electroporation notably according to the method described in the article by FROMM et al. (Nature, 319, 791-793, 1986);

by using a gene gun allowing the projection, at a very high speed, of metal particles coated with the DNA sequences of interest, thus delivering genes inside the cell nucleus, in particular according to the technique described in FIG. article by FINER et al. (Plant Cell Report, 11, 323-328, 1992);

by cytoplasmic or nuclear microinjection.

Agrobacterium tumefaciens can also be used, in particular according to the methods described in the articles by BEVAN et al. (Nucleic Acid Research, 11, 369-385, 1984) and AN et al. (Plant Phydiol., 81, 86-91, 1986), or Agrobacterium rhizogenes, in particular according to the method described in the article by JOUANIN et al. (Plant Sci., 53, 53-63, 1987). For example, plant cell transformation can be effected by transferring the T region of the Agrobacterium tumefaciens Ti tumor-inducing extrachromosomal circular plasmid, using a binary system (WATSON et al., Ed. De Boeck University, 273-292, 1994). Agrobacterium tumefaciens can also be used on whole plants, for example by depositing, at the level of the wound of a monocotyledonous plant, the bacterium harboring the DNA to be transferred, in the presence of substances released at the level of the wound of a plant. dicotyledonous. The present invention also relates to a plant cell comprising an expression cassette as defined above or a recombinant vector as defined above. The subject of the present invention is also the plants which can be obtained by a process according to the invention, with the exception of A. thaliana mutants SALK_016748 and SALK_051892. Said plants may carry mutations inhibiting laccase LAC4 and LA17 as defined above or express one or more polynucleotides capable of inhibiting the expression of said laccase LAC4 and LA17 as defined above. Of course, the present invention includes descendants, including hybrids derived from a cross involving at least one plant according to the invention, obtained by sowing or vegetative propagation, plants directly obtained by the method of the invention.

Plant material, such as protoplasts, cells, calli, leaves, stems, roots, flowers, fruits, cuttings and / or seeds, obtained from the plants in accordance with the invention (with the exception of A. thaliana mutants). SALK_016748 and SALK_051892) is also part of the object of the present invention.

The present invention also relates to the use of plants according to the invention or plant material obtained from said plants for the production of forage plants, biofuels or pulp.

The present invention will be better understood with the aid of the additional description which follows, which refers to non-limiting examples illustrating the reduction of the lignin content and the increase of the cellulolysis of the walls of a plant whose Expression of the LAC17 and / or LAC4 laccases is inhibited, as well as the appended figures:

Figure 1: agarose gel electrophoresis analysis 1% PCR products obtained from DNA genomic lines SALK_016748 (lacll mutant) (Figure

A), SALK_051892 (Iac4 mutant) (Figure B) and SALK_025690

(mutant Iac2) of A. thaliana (Figure C). A: Well 1: IKb + size marker (Invitrogen); Well 2: Tubulin from the CoIO wild line; Well 3: laccase 17 of the wild line; Wells 4 and 6: tubulin of 2 plants of line SALK_016748 (lacll); Wells 5 and 7: laccase 17 of 2 plants of the SALK__016748 (lacll) line. B: Well 1: IKb + size marker (Invitrogen), Well 2: empty; Well 3: tubulin the wild line CoIO; Well 4: empty; Wells 5 to 7: tubulin of 3 plants of line SALK__051892 (laced); Well 8: empty; Well 9: laccase 4 of the CoIO wild line; Well 10: empty; Wells 11 to 13: laccase 4 of 3 plants of the line SALK_051892 (Iac4). C: Wells 1 and 12: IKb + size marker

(Invitrogen); Wells 2 to 6 and 11: empty; Well 7: laccase

2 of the CoIO wild-type (cDNA) line; Well 8: laccase 2 of 1 plant of the CoIO wild-type (gDNA) line; Well 9: laccase

2 of 1 plant of the line SALK_025690; Well 10: control, amplified laccase 2 on plant RNA of the wild-type CoIO line having undergone the same treatment as the other plants, except that during the retro-transcription step, the reverse transcriptase was not added; Figure 2: analysis of the expression profile of laccases (LAC 2, 4, 5, 6, 10, 11, 12 and 17) of A. thaliana by electrophoresis on 1% agarose gel in TAE buffer of the products of RT-PCR obtained from cDNA cells of the floral stalk of the wild line CoIO, the line SALK_051892 {Iac4) and the line SALK_016748 {lacll). Tub = tubulin;

Figure 3: observation under optical microscope (200X magnification) of cross sections of 70 microns thick primary stem of 20 cm stained with phloroglucinol-HCl, from the wild line CoIO and the line SALK 016748 (lac11); Figure 4: Agarose gel electrophoresis analysis 1% of PCR products obtained from genomic DNA of a double mutant of A. thaliana Kim (Iac4 / lac17). Well 1: IKb + size marker (Invitrogen); Well 2: tubulin from the wild line; Well 3: tubulin of mutant Kim; Well 4: laccase 4 of the wild line; Well 5: laccase 4 of the Kim mutant; Well 6: laccase 17 of the wild line; Well 7: laccase 17 of the mutant Kim. EXAMPLE 1: SELECTION, GENOTYPING AND

CHARACTERIZATION OF MUTANTS Iacl7, Iac4 AND Iac2 O'ARABIDOPSIS THAiIANA

1) Selection of A. thaliana laccase mutants A. thaliana strains of the SALK collection in the CoIO accession showing T - DNA insertions at the LACl 7, LAC4 and LAC2 genes (respectively SALK_016748, SALK_051892 and SALK_025690) have been identified and characterized.

Mutant SALK_016748 [lac11] contains two inverted Tandem T-DNAs in the LB17 gene promoter, 146 base pairs of the ATG initiation codon.

Mutant SALK_051892 (lad) contains T-DNA inserted into the promoter of the gene encoding LAC4 at 127 base pairs of the ATG initiation codon.

The mutant SALK_025690 (Iac2) contains a T-DNA inserted into its coding sequence.

2) Genotyping of mutants lac11 (SALK 016748), Iac4 (SALK 051892) and Iac2 (SALK 025690) of A. thaliana a) Materials and Methods

Primers for genes LACIl ₁ LAC4 and LAC2 were defined using the TRACE 4 software (National Biosciences Inc., Plymouth, USA). Their sequences (5 '->3') are represented in Table 1 below: Table 1:

Sequence of primer Sequence of primer senΣ antisens

Primers for Lac 17 FST dir: Lac 17 FST rev: TCG AAG AGG GTC AAA TCT TAG CCA TGA AAT amplification of the LACl 7 GAG TT gene (SEQ ID NO: GTG AGC (SEQ ID NO:

16) 17)

Primers for irxl2 FST dir irxl2 FST

Amplification of GTG TAA GCA AAT TGG CTT GCT TGA GCA gene LAC4 CGG CAC (SEQ ID NO: TAA TCT (SEQ ID NO:

18) 19)

Primers for Lake 2 RT dir: Lake 2 RT rev _:

GCA AGA CAA AAA CAA GAA ATC TGA GGG TGG amplification of the LAC2 TCG TGA gene (SEQ ID NO: AGG AAG (SEQ ID NO:

20) 21)

The plant DNA was extracted according to the protocol described by EDWARDS et al. (Nucleic Acid Research, 19, 1349, 1991).

The PCRs were carried out in 25 μl on 30 ng of genomic DNA, with 2 mM MgCl 2, 0.4 mM of each dNTP, 0.4 mM of each primer, 1.25 units of Taq DNA polymerase (Invitrogen). PCR cycles for genotyping lacII mutants were (95 ° C 30 sec, 50 ^° C dry, 72 ° C 1 min) 28 times, with a final extension of 10 min at 72 ° C.

PCR cycles for genotyping laced mutants were (95 ° C dry, 58 ° C dry, 72 ^° C dry) 30 times, with a final extension of 10 min at 72 ° C.

The PCR cycles for genotyping the Iac2 mutants are (95 ° C dry, 54 ° C dry, 72 ° C 1 min 30 sec) 30 times, with a final extension of 10 min at 72 ° C.

The PCR products are then separated on 1% agarose gels in TAE buffer. b) Results

2 plants of the line SALK_016748, 3 plants of the line SALK_051892 and 1 plant of the line SALK_025690 were tested. The genotyping results are shown in Figure 1:

the 2 plants of the line SALK_016748 tested are homozygous for the mutation in the LACl7 gene: the presence of T-DNAs on the two coding strands of this gene prevents the amplification of a fragment of the LACl7 gene (FIG. ;

the 3 plants of the line SALK_051892 tested are homozygous for the mutation in the LAC4 gene: the presence of the T-DNA on the two coding strands of this gene prevents the amplification of a fragment of the LAC4 gene (FIG. ;

the plant of the line SALK__025690 tested is homozygous for the mutation in the LAC2 gene: the presence of T-DNA on the two strands coding for this gene prevents the amplification of a fragment of the LAC2 gene (FIG. 1C).

3) Expression of laccases in lacll and Iac4 mutants

The expression pattern of Arabldopsis laccases expressed in the floral stalk in the wild line (Columbia), the mutant lac11 (SALK_016748) and the laced mutant (SALK_051892) was determined by RT-PCR. a) Materials and Methods

A 26-cycle RT-PCR was performed on

CDNAs obtained from 1 μg of RNA extracted using an RNeasy kit (Qiagen), treated with a DNase and retro-transcribed with the SSRTII reverse transcriptase

(Invitrogen).

The primers used are described in Table 2 below: The so-called "FST" (Flanking Sequence Tag) primer pairs have been drawn on either side of the T-DNA; they were used to amplify on DNA (genomic). The so-called "RT" primer pairs have been defined in the coding sequence and make it possible to amplify on cDNAs.

The RT-PCR cycles on the lac11, Iac4 and wild-type mutants are (95 ° C. 30 sec, 50 ^° C. dry, 72 ° C. 1 min 30) 26 times, with a final extension of 10 min at 72 ° C. for laccases 2, 4, 6, 12, 17 and tubulins.

The RT-PCR cycles on the lacI _r Iac4 mutants and the wild-type line are (95 ° C. 30 sec, 55 ° C. 30 sec, 72 ° C. 1 min 30) 26 times, with a final extension of 10 min at 72 ° C. for laccases 5 and 10. The RT-PCR cycles on lac11, Iac4 and wild-type mutants are (95 ° C 30 sec, 58 ° C dry, 72 ° C 1 min 30) 26 times, with a final extension 10 min at 72 ⁰ C for laccase 11.

The RT-PCR products are then separated on 1% agarose gels in TAE buffer to visualize a difference in intensity of the amplified fragments. b) Results

The results are shown in FIG. 2. Only the level of the transcripts of laccase 17 (LAC17) decreases in the mutant lac11 and that of laccase 4 (LAC4) in the mutant lad. The mutants do not overexpress any other laccase in order to compensate for the loss of expression of LAC17 and LAC4.

4) Cytological Analysis of the Lacll Mutant a) Material and Methods

Primary stem sections of 20 cm were stained with phloroglucinol-HCl according to the protocol described by SIBOUT et al. (Plant Cell, 17, 2059-2076, 2005). The red coloration observed corresponds to the lignified cell walls. b) Results

The results of the cytological observation are shown in FIG. 3. A delay and / or a decrease in the amount of lignin deposited on the cell walls is observed in the lac11 mutant but not in the wild line (FIG. 3).

EXAMPLE 2 Obtaining and Molecular Characterization of a Double MUTANT Iacl7 / lac4 from ARABJDOPSIS THAiIANA a) Materials and Methods

Plants of line SALK_016748 (lac11) were crossed with plants of line SALK_051892 (Iac4) to obtain a double mutant Iac4 / lac17 (hereinafter referred to as Kim mutant). The double mutant Iac4 / lac17 was then characterized by genotyping following the protocol described in Example 1a) and using primers Iac4 FST dir, Iac4 FST rev, irx12 FST dir and irx12 FST rev. b) Results The genotyping results for the Kim mutant are shown in Figure 4: the Kim mutant is homozygous for mutations in the LACl1 and LAC4 genes. In fact, the presence of T-DNAs on the two coding strands of these genes prevents the amplification of a fragment of the LACl1 and LAC4 genes.

The presence of two T-DNAs in the LAC1 gene promoter and T-DNA in the LAC4 gene promoter was confirmed by amplification of sequences adjacent to T-DNAs and sequencing of amplicons. EXAMPLE 3: ANALYSIS OF THE LIGNIN CONTENT AND

CELLULOLYSIS OF SIMPLE MUTANTS Iacl7 AND Iac4 AND DOUBLE MUTANT Iacl7 / lac4 O 'ARABIDOPSIS THALIANA a) Material and Methods i) Determination of lignin content The lignin assay was carried out on the stems collected at maturity, crushed and subjected to at exhaustive extraction with ethanol / toluene (2/1, v / v) solvents, ethanol, then water; extractions performed in a Soxhlet apparatus. The extracted and dried material represents the "parietal residue" or RP (because it consists of plant walls). Elimination of soluble compounds by solvent extraction is essential before any lignin assay (these compounds may interfere with gravimetric or spectrometric assays).

The lignin content was measured by the determination of the acid-insoluble lignin fraction present in the RP and called Klason lignin (LK). This LK fraction, gravimetrically measured by treating the parietal residue with concentrated sulfuric acid (which allows the polysaccharides to be hydrolysed and a LK residue to be rinsed, dried and weighed), represents most of the parietal lignins. However, a very small fraction of the lignins can be solubilized during the treatment with sulfuric acid: it is the fraction called acid-soluble lignin (LAS) which is evaluated by measuring the absorbance of the sulfuric supernatant in the ultraviolet.

These measurements were carried out using the T222 om-83 method, known to those skilled in the art, and developed for wood and its derivatives by TAPPI (Technical Association of Pulp and Paper Industry) (DENCE, CW. The determination of lignin in: LIN, SY and DENCE, CW (Eds.), Methods in Lignin Chemistry, Springer-Verlag, pp. 33-61, 1992). ii) Study of lignin structure

The lignin structure was evaluated by thioacidolysis. The thioacidolysis of lignins liberates H, G or S thioethylated monomeric products from p-hydroxyphenyl (H), guaiacyl (G) or syringyl (S) units bound only by β-O-4 bonds (major interunit linkages in lignins natives) . These products were analyzed by gas chromatography-mass spectrometry (GC-MS) of their trimethylsilyl derivatives (TMS). Monomers trimethylsilyl H, G or S were assayed from chromatograms reconstructed respectively on ions 239, 269 or 299 (ions the most intense of their mass spectrum obtained in electronic impact). The protocol that has been employed is similar to that described by LAPIERRE et al. (Res Chem Interm, 21, 397-412, 1995) and by MIR DERIKVAND et al. (Planta 227, 943-956, 2008). The monomers H are most often minor (less than 1% of the total monomers) and were therefore not considered (except in the case of mutant plants affected in the formation of G and S units, or in the case of lignins of stress) . iii) Measurement of enzymatic degradability by in vitro cellulolysis The susceptibility of the parietal polysaccharides to enzymatic hydrolysis was evaluated in vitro, by subjecting the walls (i.e. the parietal residue RP) to an enzyme preparation. cellulasic and hemicellulasic. The protocol that was used is described by

HOFFMANN et al. (Plant Cell 16, 1446-1465, 2004), which is a protocol adapted from the method of REXEN (Animal Feed Science Technol., 2, 205-218, 1977). Said enzymatic preparation used was the Onozuka RIO Cellulase preparation extracted from Trichoderma viride, 096i / mg (SERVA ELECTROPHORESIS GmbHD, Germany). b) Results

The results are shown in Table 3 (showing the mean values between the 2 analytical replicates and the average difference between these repetitions, except for the percentage of RP for which only one measurement was performed) below, in which:

% RP = percentage of parietal residue. This percentage reflects the wall ratio in the dry sample (% by weight of the dry sample); it is given as an indication; % LK = Klason lignin level expressed as a% by weight of RP; em LK = mean difference between 2 independent analytical replicates of LK measurement; % LAS = acid-soluble lignin level expressed as a% by weight of the RP and measured from the absorbance at 205 nm of the sulfuric supernatant from the Klason lignin measurement (using a coefficient of absorptivity of 110 μg. cm ^"1 ); em LAS = average difference between 2 independent analytical LAS measurements; thio μmol / g LK = yield of monomers (G + S) of thioacidolysis, expressed in μmol per gram of Klason lignin (the H monomers) trace amounts are not considered.) When calculated on the basis of Klason lignin content, the total yield of thioacidolysis monomers reflects the proportion of units bound only in β-O-4 in lignins This structural information is important because it reflects the susceptibility of lignins to industrial delignification processes: thioacidolysis yield difference between 2 independent analytical thioacidolysis repeats S / G t hio = molar ratio of G and S monomers released by thioacidolysis of lignins. This ratio reflects the proportion of S units and G units in native lignins. In angiosperms, the importance of S units varies according to the stage of development (S units being deposited mainly at the end of lignification) as well as tissues (the fibers are richer in S units than the vessels). Therefore, when a mutant plant has a S / G ratio different from that of the control line (grown under the same conditions), this difference may be due to the fact that the mutation affects lignification over time (at the beginning or in the end) or in a tissue-specific way (affects the lignification of the fibers or that of the vessels). It is also possible that the mutation more specifically affects an enzyme involved in the biosynthesis of the coniferyl alcohol (precursor of the G units) or of the sinapyl alcohol (precursor of the S units); em S / G = mean deviation of the S / G molar ratio of thioacidolysis between 2 independent analytical thioacidolysis repeats; % loss by cellulolysis = weight loss (in

%) of RP treated with a commercial preparation of cellulase and hemicellulase enzymes; e-m cellulolysis = average difference between 2 independent analytical cellulolytic repeats; the Kim 1 and Kim 2 mutants are 2 biological repeats of the same line. Kim 1 and Kim 2 were grown on 2 separate culture trays, which may explain the variations between these replicates.

It should be noted that the mutant SALK 025690 (Iac2) does not show a decrease in the level of lignins relative to the wild line (CoIO).

Table 3: Lignin study of wild-type (CoIO) and mutant strains for laccases 4 and / or 17.

K>

From the above results it can be seen that: the homogeneity of the RP levels (60.3 to 62.6%) indicates that the mutations do not affect the rate of lignocellulosic walls of the mature stems. As mutant lines do not show a reduction in size, this suggests that mutant plants have the same productivity in terms of lignocellulosic biomass recoverable in fiber or biofuel for example;

- On the other hand, all the mutants have a Klason lignin level which is significantly lower than that of the control: the decrease is moderate for the simple mutants (decrease of 8 and 6% for the mutants Iac4 and lac11 respectively) and more marked for the double mutants. (about 19%). This decrease in lignin content, which does not affect the growth and development of the plant, is sought in the context of chemical pulp production from angiosperm lignocelluloses. It also facilitates enzymatic hydrolysis, lignins acting as barriers between enzymes and polysaccharides;

the level of acid-soluble lignin (LAS) of the mutant lines is lower than the level of LAS of the wild line. The reduced Klason lignin content (or acid-insoluble lignin) is therefore not offset by an increase in acid-soluble lignin;

the thioacidolysis yields, calculated on the basis of the LK level, are close between the wild line and the mutant lines. This result indicates that the lignins of the mutant lines contain as many labile bonds as the lignins of the wild lines. The mutations therefore did not accentuate the frequency of the inter-unit resistant bonds, which would be unfavorable in view of the production of pulp by chemical means for example;

- On the other hand, single and double mutants with the lacII mutation have a higher S / G ratio compared to that of the wild line. This result suggests that the lacII mutation could affect the onset of lignification (and thus the deposition of G units) and / or the lignification of vessels (richer in G units than fibers); the cellulolytic yield of the mutant lines is increased relative to that of the wild line. Decreasing the level of lignin Klason thus improves the efficiency of cellulolysis. EXAMPLE 4: OBTAINING TRANGENIC PEPPERS

OVER-EXPRESSING A MICRO-RNA CAPABLE OF INHIBITING THE EXPRESSION OF LAC17 AND LAC4 LACCASES

Two genetic constructs are made to over-express a microRNA (called miR397, SEQ ID NO: 77) capable of inhibiting the expression of poplar LAC17 and LAC4 laccases (Populus), under the control of either the constitutive promoter 2x35S of the CaMV is the "lignin-specific" promoter of Eucalyptus cinnamyl alcohol dehydrogenase 2 (CAD2) (referred to as EuCAD). Gateway binary vector pMDC32 (CURTIS and

GROSSNIKLAUS, Plant Physiology, 133, 462-469, 2003) is used to over-express transgenes under the control of the constitutive 2x35S promoter.

For expression under the control of the EuCAD promoter, the 2x35S promoter is excised from the above plasmid pMDC32 by digestion with HindIII-KpnI enzymes (unique restriction sites) and replaced with the EuCAD "lignin-specific" promoter.

The genetic transformation of the poplar is carried out according to the method described in LEPLE et al. (Plant Cell Rep. 11, 137-141, 1992), ie by co-cultivation of poplar stem explants with agrobacteria containing a binary vector for the expression of miR397, isolation of transgenic calli and regeneration of transformed seedlings.

Transgenic calli are selected for the regeneration step. Seedlings are regenerated at from these different calluses, thus corresponding to different transformation events. Each seedling is cloned by multiplication.

One copy of each transgenic line is used for: the study of lignin structure by thioacidolysis (see above) on a stem fragment; to identify spatial variations in the amount of lignins, by infra-red imaging in FTIR-ATR ("Fourier Transformed! InfraRed Spectroscopy - Attenuated Total Reflectance") and histochemical staining with phloroglucinol.

These first phenotyping analyzes identify lines with reductions in the amount of lignin in the wood.

Lines are then analyzed for the expression of transgenes and genes coding for LAC17 and LAC4. These expression analyzes are carried out by quantitative RT-PCR (qRT-PCR). EXAMPLE 5: OBTAINING BRACHYPODIUM

TRANGENIC DISTACHYON OVER-EXPRESSING MICRO-RNA CAPABLE OF INHIBITING THE EXPRESSION OF LAC17 AND LAC4 LACCASES

Gateway compatible and specific binary vectors for the transformation of monocotyledons, containing a sequence coding a micro-RNA of sequence SEQ

ID NO: 78 or 79, under the control of either a constitutive promoter, for example the maize ubiquitin promoter (ZmUbi), or a "specific lignin" promoter, and a selection gene such as the gene pat (conferring resistance to basta), are used for the genetic transformation of Brachypodium distachyon.

The analysis of the structure and lignin content of the transgenic plants obtained can be performed using the same methods as for the analysis of the transgenic poplars above. EXAMPLE 6 PRODUCTION OF TRANGENIC CORN SURPRISING A MICRO-RNA CAPABLE OF INHIBITING THE EXPRESSION OF LACC17 AND LAC4 LACCASES

A vector as described for the genetic transformation of Brachypodium can be used for the genetic transformation of maize.

It is also possible to use the integrative vector L1038 (represented by the sequence SEQ ID NO: 80), which contains an expression cassette comprising a gene for resistance to a herbicide (Basta resistance gene), an expression cassette comprising a gene encoding a fluorescent protein to track the transgene without genotyping (gene encoding a GFP under the control of a Actin promoter), a "Gateway triple" cassette attR4-ccdB-attR3 (where attR4 and attR3 are recombination sites and ccdB is a negative selection gene), which makes it possible to recombine a promoter of choice (attL4-attR1 ends), a gene of choice (attL1-attL2 ends) (in this case a microRNA miR397) and a mock (attR1 -attL3) (see the User Manual published by Invitrogen, "MultiSite Gateway Pro", Version B, October 3, 2006).

Claims

1) Process for reducing the lignin content of a plant and increasing the cellulolysis of the walls of said plant, characterized in that the expression and / or the activity in said plant is totally or partially inhibited: a) a laccase of which the polypeptide sequence has at least 60% identity or at least 75% similarity to the sequence SEQ ID NO: 2, and comprises, from its N-terminus to its C-terminus, at least one one of the 4 consensus peptide domains i) to iv) respectively, following: i) the consensus peptide domain of sequence SEQ ID NO: 12 or 38, ii) the consensus peptide domain of sequence SEQ ID NO: 13 or 39, iii) the consensus peptide domain of sequence SEQ ID NO: 14 or 40, iv) the sequence consensus peptide domain

SEQ ID NO: 15 or 41, and b) a laccase of which the polypeptide sequence has at least 65% identity with the sequence SEQ ID NO: 4. 2) Method according to claim 1, characterized in that total or partial inhibition of the expression and / or the activity in said plant of said laccases is obtained by mutagenesis of the gene coding for these laccases, or by inhibition or modification of the transcription or translation of these laccases.

3) Method according to claim 1 or claim 2, characterized in that said laccase as defined in paragraph a) is: in corn (Zea mays), chosen from the peptide sequences SEQ ID NO: 5, 6 and 42 to 47, preferably SEQ ID NO: 5, 6, 42, 43, 44 and 45, in sugar cane (Saccharum officinarum), the sequence SEQ ID NO: 7, in sorghum (Sorghum bicolor), chosen from the peptide sequences SEQ ID NO: 8 to 11, in Brachypodium, chosen from the peptide sequences SEQ ID NO : 48 to 55, preferably the sequences SEQ ID NO: 50 and 51, in rice, chosen from the peptide sequences SEQ ID NO: 56 to 62, preferably the sequence SEQ ID NO: 58, and in the poplar, chosen among, the peptide sequences SEQ ID NO: 63 to 68, preferably the sequences SEQ ID NO: 63, 67 and 68.

4) Method according to any one of claims 1 to 3, characterized in that said laccase as defined in paragraph b) is: in Brachypodium, the peptide sequence SEQ ID NO: 69, in rice, the peptide sequence SEQ ID NO: 70, and in poplar, selected from the peptide sequences SEQ ID NO: 71 to 74.

5) Construction of recombinant DNA, characterized in that it comprises one or more polynucleotides capable of inhibiting the expression of a laccase whose polypeptide sequence has at least 60% identity or at least 75% similarity with the sequence SEQ ID NO: 2 and comprises, from its N-terminal end towards its C-terminal end, at least one of the four consensus peptide domains i) to iv) respectively, as follows: i) the peptide sequence consensus domain SEQ ID NO: 12 or 38, ii) the consensus peptide domain of sequence SEQ ID NO: 13 or 39, iii) the consensus peptide domain of sequence SEQ ID NO: 14 or 40, iv) the consensus peptide domain of sequence SEQ ID NO: 15 or 41, and a laccase whose polypeptide sequence has at least 65% identity with the sequence SEQ ID NO: 4.

6) DNA construct according to claim 5, characterized in that said polynucleotide encodes an antisense RNA, an interfering RNA, a microRNA or a ribozyme targeting genes encoding said laccases. 7) expression cassette, characterized in that it comprises one or more DNA constructs as defined in claims 5 or 6, under transcriptional control of a suitable promoter.

8) Recombinant vector, characterized in that it comprises one or more DNA constructs as defined in claims 5 or 6, or an expression cassette as defined in claim 7.

9) plant cell, characterized in that it comprises an expression cassette as defined in claim 7 or a recombinant vector as defined in claim 8.

10) Plant obtainable by the method according to any one of claims 1 to 4, with the exception of the lines of Arabidopsis thaliana SALK_016748 and SALK_051892.

11) Use of a plant obtainable by the method according to any one of claims 1 to 4, or plant material obtained from said plant, for the production of forage plants, biofuel or pulp. paper.