BR102017023837A2 - METHODS OF AMENDMENT OF THE STRUCTURE OF THE CELLULAR PLANT WALL - Google Patents

Abstract

methods of altering plant cell wall structure. The present invention belongs to the field of plant genetics and describes a new and inventive method of producing plants with altered cell wall structure. The invention further provides nucleic acid molecules, constructs and other agents associated with the manipulation of plant cell wall structure genes. more specifically, the invention relates to gene constructs aimed at suppressing expression of acyl transferase encoding genes, combined with tissue-specific and constitutive promoters. Also provided is the plant incorporating such elements, as well as method of altering plant cell wall structure using molecules in different forms of presentation.

Description

METHODS OF CHANGING THE STRUCTURE OF THE PLANT CELL WALL.

FIELD OF THE INVENTION [1] The present invention belongs to the field of plant genetics and relates to nucleic acid molecules, constructs and other agents associated with the manipulation of plant cell wall structure genes. More specifically, the invention is related to gene constructs that aim at suppressing the expression of genes encoding acyl transferases, combined with tissue-specific and constitutive promoters. In particular, the invention relates to the genes responsible for the xylan feruloylation in grasses called BAHD1, BAHD3, BAHD5 and BAHD8, present in species of the Poaceae family, such as Setaria viridis, Brachypodium distachyon and Saccharum officinarum. This invention describes the use of such gene constructs in transgenic plants to reduce the levels of the arabinoseferuloyl transferase transcripts, which will produce lesser amount of ferulates bound to the cell wall, reducing the crosslinking between its polymers. Alternative approaches that could be used to decrease the transcription levels of the BAHD1, BAHD3, BAHD5 and BAHD8 genes, or the activity of the enzymes encoded by these genes, are also described.

DESCRIPTION OF THE STATUS OF THE TECHNIQUE [2] The family Poaceae (grasses) is composed of a group of plants of which maize, sorghum, wheat, Miscanthus, Panicum spp., Brachypodium spp., Setaria spp., Brachiaria spp., rice, sugar cane, among others. Species belonging to this family have a typical cell wall architecture that distinguishes them from other plant groups. Most plants have xyloglucan as the main hemicellulose, while grasses have glucuronoarabinoxylans (GAXs) as main hemicellulose (Saavedra, F .; Karácsonyi, S; Alfôldi, J. Studies of the polysaccharides of sugarcane (Saccharum officinarum): structural features of the

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2/36 waterinsoluble D-xylans. Carbohydrate Research, v. 180, η. 1, p. 61-71, 1988; Souza, AP. Sugarcane and climate change: effects of a CO2-enriched atmosphere on the growth, development and metabolism of Saccharum ssp. 2007. Dissertation (Master in Cellular and Structural Biology) State University of Campinas, Institute of Biology, Campinas, 2007), although it also has, in small proportions, xyloglucans and mannans. In addition to GAXs, β-glucans are relatively abundant in all grass tissues.

[3] A key difference between the cell wall of grasses and other plants is the presence of hydroxycinnamic acids (HCAs) ester linked to GAX arabinose. These HCAs are composed of ferulic acid (FA) and para-cumaric acid (pCA). AF residues esterified to proximal GAX may undergo dimerization, linking the polysaccharides with each other. The presence of ferulic acid gives the grass wall special resistance to UV rays and to the attack of hydrolytic enzymes of pathogens (Oliveira, DM et al. Ferulic acid: a key component in grass lignocellulose recalcitrance to hydrolysis. Plant Biotechnology Joumal, v. 13, No. 9, pp. 1224-1232, 2015).

[4] Grass cell walls are characterized by having a large amount of esterified ferulates, as mentioned above. The grouping mode appears to be highly conserved, in which ferulic acid is attached to the first hydroxyl formed at the C5 position when arabinose is in the form of furanose via an ester bond. The chemical characterization of cell wall fragments resulting from partial enzymatic hydrolysis revealed oligosaccharide ferulate structures as follows: FA-Ara-Xil; FA-Ara- (Xil) 2; FA-Ara- (Xil) 3 (Hatfield, RD; Ralph, J; Grabber, JH. Cell wall cross-linking by ferulates and diferulates in grasses. Joumal of the Science Food and Agriculture, v. 79, p. 403-407 , 1999).

[5] AF residues bind through oxidation reactions to form a variety of dehydrodiferulate dimers, making the cross-linking of hemicellulose chains (Ralph, J, Quideau, S; Grabber, JH; Hatfield, RD. Identification and synthesis of new ferulic acid dehydrodimers present in grass cell walls. Joumal of the Chemical Society, Perkin Transactions 1, v. 23, p. 3485-3498, 1994 .; Oliveira, DM et

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3/36 al. Ferulic acid: a key component in grass lignocellulose recalcitrance to hydrolysis. Plant Biotechnology Joumal, v. 13, n. 9, p. 1224-1232, 2015). Ferulic acid is easily oxidized by peroxidase and, as products of coupling reactions via free radical, a wide variety of differulates (Iiyama, K; Lam, TBT; Stone, BA. Covalent cross-links in the cell wall. Physiology, v. 104, pp. 315-320, 1994; Hatfield, RD; Ralph, J; Grabber, JH.Cell wall cross-linking by ferulates and diferulates in grasses. Joumal of the Science Food and Agriculture, v. 79, 403-407, 1999), which are responsible for 70% of the total ferulates, and can be found in the form of 5-5, 8-8, 8-5 and 8-0-4 differulates.

[6] GAX feruloylation is important because it directly links xylans. In addition, esterified ferulates are incorporated into lignin through ether and carbon-carbon bonds, as nucleation sites for the formation of lignin. GAX in grasses play an important structural role in linking cellulose microfibrils. Feruloylation is also responsible for binding lignin to the cellulose / xylan network through the lignin-ferulate-xylan complexes (Jacquet, G; Pollet, B; Lapierre, C; Mhamdi, F; Ronaldo, C. New ether-linked ferulic acid-coniferyl alcohol dimers identified (Joumal of Agricultural and Food Chemistry, v. 43, n. 10, p. 2746-2751, 1995). The ester-ether ferulic acid bonds formed between lignin and GAX are attributed to an important role in limiting cell wall hydrolysis in grasses.

[7] Ferulic acid esterified to GAX in grasses interferes with the digestion of the cell wall, preventing the binding of enzymes, such as xylanase to its substrate, which is essential for the hydrolysis stage. Therefore, the reduction in the cross-linking level of cell wall carbohydrates may represent an improvement in the speed and extent of cell wall digestibility in grasses and their bioconversion into ethanol and nutrients via animal feed (Dos Santos, WD; Ferrarese, MML ; Ferrarese-Filho, O. Ferulic acid: an allelochemical troublemaker. Functional Plant Science and Biotechnology, v. 2, η. 1, p. 47-55, 2008; Hatfield, RD. Cell wall polysaccharide interactions and degradability. In: Jung , HG et al (Eds.), Forage Cell Wall Structure and Digestibility, ASA-CSSA-SSSA, Madison: WI, 1993. pp. 285-313;

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Grabber, J H; Ralph, J; Hatfield, RD. Cross-linking of maize walls by ferulate dimerization and incorporation into lignin. Journal of Agricultural and Food Chemistry, v. 48, n. 12, p. 6106-6113, 2000 .; Yu, ANQ. et al. The institute for genome research Osal rice genome annotation database. Plant Physiology, 138: 18-26, 2005; Buanafina, MMO et al. Manipulating the phenolic acid content and digestibility of Italian ryegrass (Lolium multtflorum) by vacuolar-targeted expression of a fungai ferulic acid esterase. Applied Biochemistry and Biotechnology, v. 130, η. 1, p. 416-426, 2006; Cao, BB et al. Microbial release of ferulic and p-coumaric acids from forages and their digestibility in lactating cows fed total mixed rations with different forage combinations. Journal of the Science of Food and Agriculture, v. 96, n. 2, p. 650-655, 2016).

[8] The BAHD enzyme group is divided into five subtypes (D'Auria, JC. Acyltransferases in plants: a good time to be BAHD. Current Opinion in Plant Biology, v. 9, p. 331-340, 2006), although other subclades have been proposed (Touminen, LK et al., Differential phylogenetic expansions in BAHD acyltransferases across five angiosperm taxa and evidence of divergent expression among Populus paralogues. BMC Genomics, v. 12, η. 1, p. 236, 2011) . Several characterized members use hydroxycinnamyl-CoAs as substrates, including hydroxycinnamyl-CoA ωchiquimate / hydroxycinnamyl transferase (HCT) involved in the synthesis of lignin precursors (Hoffmann, L et al. Purification, cloning, and properties of an acyltransferase controlling shikimate and quinate ester intermediates in phenylpropanoid metabolism (The Journal of Biological Chemistry, v. 278, p. 95-103, 2003). Other works describe the suberins and cutins feruloil transferases of Arabidopsis thaliana acting on the transfer of acyl group acceptors of hydroxycinnamyl-CoA 03-hydroxy fatty acids (Molina, I et al. Identification of an Arabidopsis feruloyl-coenzyme A transferase required for suberin synthesis Plant Physiology, v. 151, p. 1317-1328, 2009; Rautengarden C et al. The interconversion of UDP-arabinopyranose and UDP-arabinofuranose is indispensable for plant development in Arabidopsis. The Plant Cell, v. 23, n. 4 , pp. 1373-1390, 2011.).

[9] In Bartley et al. Overexpression of a BAHD acyltransferase, OsAtlO, alters rice cell wall hydroxycinnamic acid content and saccharification. Plant Physiology, v.

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161, ρ. 1615-1633 (2013) it is shown that rice strains overexpressing the OsATlO gene resulted in an increase in pCA, involved in the modification of glucuronoarabinoxylan, showed an increase of up to 40% in saccharification. Buanafina et al. Functional testing of a PF02458 homologue of putative rice arabinoxylan feruloyl transferase genes in Brachypodium distachyon. Plant, v. 243, p. 659-674 (2016) reported effects on changes in the expression of Bradi2g43520 (BdATT, BdBAHD5 in our nomenclature), a homologue of the LOC_Os01g42880 rice gene in Brachypodium distachyon. Gene silencing in several independent RNAi :: BdATl strains resulted in a reduction of up to 35% in the levels of ferulic acid in leaves and stems compared to control plants.

[10] There is a need to increase digestibility in grasses both for the production of biofuel via second generation ethanol (2G) and for the production of animal feed, in addition to the production of chemical compounds with high added value. This invention meets these needs.

SUMMARY OF THE INVENTION [11] The invention provides a method for generating plants with reduced levels of ferulic acid and increased saccharification. Transgenic plants produced by this strategy can be used both for the production of biofuel via second generation ethanol (2G) and for the production of animal feed.

[12] In one aspect, the invention provides a method of producing a plant with altered cell wall structure by inserting into said plant a DNA molecule whose expression results in an RNA molecule whose sequence comprises at least one segment of 18 or more contiguous nucleotides complementary to at least one fragment of SEQ ID No 1, SEQ ID No 3, SEQ ID No 5 or SEQ ID No 7. The invention also provides for the plant and / or parts of the plant produced by said method.

[13] In another aspect, the invention provides an isolated nucleic acid molecule comprising a nucleic acid sequence with at least 90, 95, 99 or 100% similarity to the sequence described in SEQ ID No 9, SEQ ID No 10, SEQ ID No

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6/36 or in SEQ ID No 12. An isolated nucleic acid molecule comprising a nucleic acid sequence with at least 90, 95, 99 or up to 100% similarity to the complement of the sequence described in SEQ ID No 9 , SEQ ID NO 10, SEQ ID NO 11 or SEQ ID NO 12.

[14] A further aspect of the invention provides a chimeric gene that comprises at least one polynucleotide comprising a nucleic acid sequence with at least 90, 95, 99 or 100% similarity to the sequence described in SEQ ID No 9, SEQ ID No 10, SEQ ID NO 11 or SEQ ID NO 12 operationally linked to an active promoter. Also discussed is a gene construct comprising one or more of said chimeric gene.

[15] The invention further provides for a gene construct that comprises a first region with a sequence of 18 or more consecutive nucleotides having at least 90, 95, 99 or up to 100% similarity with at least one sequence of 18 or more consecutive nucleotides from sense nucleotide sequence selected from SEQ ID No 9, SEQ ID No 10, SEQ ID No 11 and SEQ ID No 12 and a second region with a nucleotide sequence of 18 or more consecutive nucleotides having at least 90, 95, 99 or even 100% sequence similarity with the complement of 18 or more consecutive nucleotides from at least one nucleotide sequence selected from SEQ ID No 9, SEQ ID No 10, SEQ ID No 11 and SEQ ID No 12.

[16] In another aspect, a method is provided for producing a transgenic plant cell, plant, seed or plant part capable of expressing a dsRNA capable of suppressing the expression of the BAHD1, BAHD3, BAHD5 or BAHD8 gene, through transformation of a plant cell with a gene construct as described above.

[17] The invention further provides a vector comprising an isolated nucleic acid molecule comprising a nucleic acid sequence with at least 90, 95, 99 or 100% similarity to the sequence described in SEQ ID No 9, SEQ ID No 10 , SEQ ID NO 11 or SEQ ID NO 12, as well as the double-stranded RNA molecule produced from the expression of said vector.

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7/36 [18] Cell and transformed plant comprising an isolated nucleic acid molecule comprising a nucleic acid sequence with at least 90, 95, 99 or 100% similarity to the sequence described in SEQ ID NO 9, SEQ ID No 10, SEQ ID No 11 or SEQ ID No 12, as well as seed or propagating part of the plant.

[19] In another aspect, a method of altering cell wall structure by exposing a plant or part of a plant to a double-stranded RNA molecule whose sequence comprises at least a segment of 18 or more consecutive nucleotides of at least a sequence selected from SEQ ID No 9, SEQ ID No 10, SEQ ID No 11 and SEQ ID No 12.

[20] An isolated nucleic acid molecule composition comprising a nucleic acid sequence with at least 90, 95, 99 or 100% similarity to the sequence described in SEQ ID No 9, SEQ ID No 10, SEQ ID No. 11 or SEQ ID No. 12, as well as a method of producing a plant with altered cell wall structure by exposing the plant to said composition. Said plant is also provided.

BRIEF DESCRIPTION OF THE FIGURES [21] Figure 1: Schematic representation of the binary vectors for silencing via RNAi of the BAHD genes. A - Binary vector used for the transformation of S. viridis; B Binary vector used for processing sugarcane.

[22] Figure 2: Relative expression of the BAHD1, BAHD5 and BAHD8 genes of the S. viridis transgenic T0 events.

[23] Figure 3: Relative expression of the BAHD1 gene for homozygous transgenic T2 events (Ev 17.3 and Ev 18.1) from S. viridis. (A) Thatch; (B) Leaf and (C) Root. NT - unprocessed plant.

[24] Figure 4: Relative expression of S. viridis transgenic events with the BAHD1 gene under the control of the SilRX1 promoter.

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8/36 [25] Figure 5: Quantification of trans ferulic acid (TFA) and p-cumaric acid (PCA) in stem. NT - unprocessed plant; Ev. 17.3 and Ev. 18.1 - homozygous transgenic events. The letters a and b in the graph express a statistically significant difference in relation to the NT or between the events, respectively, by the one-way analysis of variance (ANOVA) followed by the Tukey test (p <0.05).

[26] Figure 6: Quantification of trans ferulic acid (TFA) and p-cumaric acid (PCA) in leaves. NT - unprocessed plant; Ev. 17.3 and Ev. 18.1 - homozygous transgenic events. The letters a and b in the graph express a statistically significant difference in relation to the NT or between the events, respectively, by the one-way analysis of variance (ANOVA) followed by the Tukey test (p <0.05).

[27] Figure 7: Quantification of trans ferulic acid (TFA) and p-cumaric acid (PCA) in roots. NT - unprocessed plant; Ev. 17.3 and Ev. 18.1 - homozygous transgenic events. The letters a and b in the graph express a statistically significant difference in relation to the NT or between the events, respectively, by the one-way analysis of variance (ANOVA) followed by the Tukey test (p <0.05).

[28] Figure 8: Lignin content in NT - unprocessed plant; Ev. 17.3 and Ev. 18.1 - homozygous transgenic events. (A) Thatch; (B) Leaves and (C) roots. The letters a and b in the graph express a statistically significant difference in relation to the NT or between the events, respectively, by the one-way analysis of variance (ANOVA) followed by the Tukey test (p <0.05).

[29] Figure 9: Quantification of the monomers that make up the cell wall of T2 BAHD1 and NT events. (A) Thatch; (B) Leaf and (C) Root.

[30] Figure 10: Saccharification of S. viridis with commercial enzymatic cocktail Cellic®Ctec3 at 10 FPU (Filter paper units) / g of biomass). NT - unprocessed plant; Ev. 17.3 and Ev. 18.1- homozygous transgenic events.

[31] Figure 11: Phenotyping of the homozygous events of S. viridis and sugar cane. (A) S. viridis biomass; (B) Weight of 1,000 seeds of S. viridis, (C) Phenotype of S. viridis and (D) Phenotype of sugar cane

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9/36 [32] Figure 12: Relative expression of transgenic sugarcane events with the BAHD1 gene.

[33] Figure 13: Quantification of trans ferulic acid (TFA) and p-cumaric acid (PCA) in leaves of sugarcane events. NT - unprocessed plant.

[34] Figure 14: Percentage of glucose released by saccharification of leaves from sugarcane events at different times of enzymatic hydrolysis (TO = Ohs; T3 = 3hs; T6 = 6hs; TIO = 10hs; T24 = 24hs). NT - unprocessed plant; Ev - transgenic events generated.

DETAILED DESCRIPTION OF THE INVENTION [35] The purpose of the present invention is to provide a method for modifying expression, as well as an effective promoter sequence for plants, preferably Poaceas, in order to make possible the production of genetically modified varieties by silencing the genes of interest. in every plant or in parts of it.

[36] In the context of this description, a number of terms will be used and will therefore be further detailed below:

[37] The terms polynucleotide and nucleic acid are used in an equivalent manner and refer to a single or double-stranded polymer of deoxyribonucleotides or ribonucleotides read from the 5 'to the 3' end. A nucleic acid of the present invention generally contains phosphodiester bonds, although in some cases nucleic acid analogs may be used which may have alternative skeletons, containing, for example, phosphoramidate, phosphorothioate, phosphorodithioate, or O-methyl phosphoramidite ( see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press); positive skeletons; nonionic skeletons, and ribose-free backbones. Thus, nucleic acids or polynucleotides can also include modified nucleotides that allow correct reading by the polymerase. Polynucleotide sequence or nucleic acid sequence includes both the sense and antisense chains of a nucleic acid as well as

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10/36 single strands or in duplex. The description of a single tape also defines the complementary sequence; thus, the sequences described here also provide the sequence complement. “Complementarity” refers to the specific pairing of purine bases and pyrimidines that consist of nucleic acids: pairs of adenine with thymine and pairs of guanine with cytosine. Then, the "complement" of a first nucleic acid fragment refers to the second nucleic acid fragment whose nucleotide sequence is complementary to the first nucleotide sequence. Unless specifically indicated, a particular nucleic acid sequence also implicitly encompasses its variants (for example, substitutions of degenerate codons) and complementary sequences, as well as the explicitly indicated sequence.

[38] In more complex organisms, deoxyribonucleic acid (DNA) is a nucleic acid that contains information about the genetic material of a given organism, while ribonucleic acid (RNA) is a nucleic acid that is involved in the transfer of DNA information in proteins. A “genome” is every major part of the genetic material contained in each cell in an organism. The term "nucleotide sequence" refers to sequences of nucleotide polymers, forming a strand of DNA or RNA, which can be single or double stranded, optionally synthetic, unnatural or with altered nucleotide bases capable of incorporation within of DNA or RNA polymers. The term "oligomer" refers to short sequences of nucleotides, usually up to 100 bases in length. The term "homologous" refers to the ancestral link between the nucleotide sequences of two nucleic acid molecules or between the amino acid sequences of two protein molecules. The estimation of such homology is provided by hybridizing DNA-DNA or RNARNA under stringent conditions as defined in the prior art (as mentioned in US20030074685, Hames, BD and Higgins, SJ (1985) Nucleic Acid Hybridization: A Practical Approach , IRL Press Oxford, UK); or by comparing sequence similarity between two nucleic acid or protein molecules (as mentioned in US20030074685, Needleman et al., J. Mol. Biol. (1970) 48: 443-453).

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11/36 [39] "Gene" refers to the nucleotide fragment that expresses a specific protein, including preceding (5 'untranslated region) and later (3' untranslated) regulatory sequences to the coding region. "Native gene" refers to an isolated gene with its own regulatory sequence found in nature. "Chimeric gene" refers to the gene that comprises coding, regulatory and heterogeneous sequences not found in nature. The chimeric gene of the present invention comprises isolated nucleic acid molecules, in the sense or antisense orientation, operably linked to active promoters. Gene constructs of the present invention may contain one or more chimeric genes and may or may not have introns. “Endogenous gene” refers to the native gene normally found in its natural location in the genome and is not isolated. An "exogenous gene" refers to a gene that is not normally found in the host organism, but that is introduced by gene transfer. "Pseudogene" refers to a nucleotide sequence that does not encode a functional enzyme.

[40] "Coding sequence" refers to the DNA sequence that encodes a specific protein and excludes the non-coding sequence. An "interrupted coding sequence" means the sequence that acts as a separator (for example, one or more introns connected through junctions). An "intron" is a nucleotide sequence that is transcribed and is present in the pre mRNA, but is removed by dividing and reconnecting the mRNA within the cell generating a mature mRNA that can be translated into a protein. Examples of introns include, but are not limited to, intron pdk, intron pdk2, intron catalase of castor, intron Delta 12 denaturase cotton, Delta 12 denaturase Arabidopsis thaliana, ubiquitin corn intron, SV40 intron, SV40 intron synthase.

[41] "RNA transcript" refers to the product resulting from transcription catalyzed by the RNA polymerase of a DNA sequence. When the RNA transcript is a perfect copy of the DNA sequence, it is referred to as a primary transcript or it can be an RNA sequence derived from a post-transcriptional process of the primary transcript and is then referred to as a mature transcript. “Messenger RNA (mRNA)” refers to RNA without introns. “RNA sense” refers to an RNA transcript that

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12/36 includes mRNA. “Antisense RNA” refers to an RNA transcript that is complementary to all parts of a primary transcript or mRNA and that can block the expression of a target gene by interfering with the processing, transport and / or translation of its primary transcript or mRNA. The complementarity of an antisense RNA can be with any part of the specific gene transcript, i.e., 5 'untranslated sequence, 3' untranslated sequence, introns or coding sequence. In addition, antisense RNA may contain regions of ribozyme sequences that increase the effectiveness of antisense RNA to block gene expression.

[42] Two nucleic acid sequences or polypeptide sequences are said to be identical if the sequence of nucleotides or amino acid residues, respectively, in the two sequences, is the same when aligned for maximum matching, as described below. The terms "identical" or percent identity, in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences and subsequences that are the same or have a specified percentage of amino acid or nucleotide residues that they are the same when compared and aligned for maximum correspondence over a comparison range, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. When the percentage of sequence identity is used in reference to proteins or peptides, it is known that the positions of residues that are not identical often differ by replacing the conserved amino acid, where the amino acid residues are replaced by other amino acid residues with chemical properties similar (for example, charge or hydrophobicity) and therefore do not alter the functional properties of the molecule. When the sequences differ in conservative substitutions, the percent identity of the sequence can be adjusted to correct the conserved nature of the substitution. The ways to make this adjustment are well known. This usually involves marking a conservative substitution as partial rather than complete incompatibility, thereby increasing the percentage of sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conserved substitution is given a zero and a conserved substitution is given a score between zero and 1. The score

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13/36 of conserved substitutions is calculated according to, for example, the algorithm of Meyers and Miller (1988), for example, from the PC / GENE program (Intelligenetics, Mountain View, Calif., USA).

[43] For sequence comparison, typically a sequence acts as a reference sequence, which the test sequences are compared to. When using a sequence comparison algorithm, tests on reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and the sequence of algorithm parameters in the program are designated. Standard program parameters can be used, or alternative parameters can be used. The comparison sequence of the algorithm then calculates the percent identity of the test sequence in relation to the reference sequence, based on the program parameters.

[44] Nucleic acid or protein sequences that are substantially identical to a reference sequence include conservatively modified variants with respect to a particular nucleic acid sequence, conservatively modified variants refer to the nucleic acids that encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence for essentially identical sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, codons GCA, GCC, GCG and GCU encode the amino acid alanine. Thus, at each position where an alanine is specified by a codon, the codon can be changed to any of the codons described above without changing the encoded polypeptide. Such variations of nucleic acid are silent variations, which are a kind of conservatively modified variant form. Each nucleic acid sequence mentioned herein that encodes a polypeptide also describes each possible silent variation of the nucleic acid. A specialist will recognize that each codon in a nucleic acid (except AUG, which is normally the only codon for methionine) can be modified to give a functionally identical molecule. So, each silent variation of

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14/36 a nucleic acid encoding a polypeptide is implied in each described sequence.

[45] As with amino acid sequences, one skilled in the art will recognize that individual substitutions in a nucleic acid, peptide, polypeptide or protein sequence that alters a single amino acid or a small percentage of amino acids in the coded sequence is a conservatively modified variant , where the changes result in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables that provide functionally similar amino acids are well known in the field.

[46] The following six groups each contain amino acids that show conservative substitutions (Voet, D; Voet, JG; Pratt, CW. Fundamentals of biochemistry: life at the molecular I took: John Wiley and Sons New York: 2013):

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[47] Another indication that the nucleotide sequences are substantially identical is whether two molecules hybridize to each other, or to a third nucleic acid, under stringent conditions. The stringent conditions are sequence dependent and vary according to the circumstances. Generally, stringent conditions are selected to be approximately 5 ° C below the denaturation point (Tm) for the specific sequence at a defined ionic strength and pH. Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence remains hybridized with a perfectly matched probe. Typically, stringent conditions will be those in which the salt concentration is 0.02 M at pH 7 and the temperature is at least about 60 ° C. For example, stringent

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15/36 hybridization, such as DNA-RNA hybridizations in a transfer technique are those that include at least one wash in 0.2 x SSC at 55 ° C for 20 minutes, or equivalent conditions.

[48] The term promoter, as used herein, refers to a polynucleotide sequence capable of directing the transcription of a DNA sequence in a cell. Thus, the promoters used in the polynucleotide constructs of the invention include elements of control of cis and irans-active transcription and regulatory sequences that are involved in the regulation or modulation of the time and / or the rate of transcription of a gene. For example, a promoter can be a czs-active transcription control element, including an enhancer, a promoter, a transcription terminator, an origin of replication, a chromosomal integration sequence, 5 'and 3' untranslated regions, or an intronic sequence, which are involved in the regulation of transcription. These czs-acting sequences normally interact with proteins or other biomolecules to perform (on / off, regulate, modulate, etc.) gene transcription. The promoters are located in the 5 'region of the transcribed gene, and as used herein, include the 5' sequence of the translation initiation codon (i.e., including the 5 'untranslated region of the mRNA, typically comprising 100 - 200 bp). In most cases, the nuclear promoter sequences are found within 1 to 2 kb of the translation initiation site, more often at 1 kb and often within 500 bp of the translation initiation site. By convention, the promoter sequence is normally in the sequence of the coding strand of the gene it controls. In the context of this application, the promoter is usually referred to by the name of the gene which it naturally controls expression. A promoter used in an expression cassette of the invention is referred to by the name of the gene. Reference to a promoter by name includes wild type, native promoter as well as variants of the promoter that retain the ability to induce expression. The reference to a promoter by name is not restricted to a specific plant species, but also covers a promoter of a corresponding gene in other plant species.

[49] A constitutive promoter in the context of this invention refers to a promoter that is capable of initiating transcription in almost all cell types, whereas a

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16/36 cell-specific or tissue-specific promoter initiates transcription only in one or a few particular types of cells or groups of cells that form a tissue. In some embodiments, a promoter is tissue specific if the levels of transcription initiated by the promoter in the cell wall are at least 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, eight times, 9 times , 10 times, 50 times, 100 times, 500 times, 1000 times higher or more, compared to levels of transcription initiated by a non-cell wall tissue promoter.

[50] A polynucleotide is heterologous to an organism or a second polynucleotide sequence if it originates from a distant species or, if it is from the same species, it is modified from its original form. For example, when a polynucleotide that encodes a polypeptide sequence is said to be operably linked to a heterologous promoter, it means that the polynucleotide sequence that encodes the polypeptide is derived from one species while the promoter sequence is derived from another different species , or if both are derived from the same species, the coding sequence is not naturally associated with the promoter, for example, it is a sequence encoded by genetic manipulation, for example, from a different gene in the same species, or an allele of a different ecotype or variety.

[51] The term operationally linked refers to a functional relationship between two or more polynucleotide segments (for example, DNA). Typically, it refers to the functional relationship of a transcriptional regulation sequence to a transcribed sequence. For example, a promoter or enhancer sequence is operably linked to a DNA or RNA sequence that stimulates or modulates the transcription of the DNA or RNA sequence in an appropriate host cell or other expression system. Generally, the promoter transcriptional regulation sequence that is operably linked to a transcribed sequence is physically contiguous to the transcribed sequence, that is, they are cis-acting. However, some transcriptional regulation sequences, such as enhancers, need not be contiguous or physically located in close proximity to the coding sequences whose transcription they increase.

Petition 870170085237, dated 11/06/2017, p. 37/79 \ Ί / 36 [52] In one aspect of the invention, the promoter is a promoter expressed in plants. As used here, the term "plant-expressed promoter" means a DNA sequence that is able to initiate and / or control transcription in a plant cell. This includes any promoter of plant origin; any promoter of non-plant origin which is capable of directing expression in a plant cell, for example promoters of viral or bacterial origin such as CaMV35S (as mentioned in patent application US20030175783, Hapster et al, 1988 Mol. Gen. Genet. 212, 182-190) and gene promoters present in Agrobacterium T-DNA, tissue-specific or organ-specific promoters, including but not limited to seed-specific promoters (WO8903887), specific promoters of primordial organs (as mentioned in patent application US20030175783, An et al., 1996 The Plant Cell 8, 15-30), stem-specific promoters (as mentioned in patent application US20030175783, Keller et al., 1988 EMBO J. 7: 3625-3633), specific leaf promoters (as mentioned in patent application US20030175783, Hudspeth et al., 1989 Plant Mol Biol 12: 579-589), specific mesophilic promoters, specific root promoters (as mentioned in patent application US2003 0175783, Keller et al., 1989 Genes Devei. 3: 1639-1646), tuber-specific promoters (as mentioned in patent application US20030175783, Keil et al., 1989 EMBO J. 8: 1323: 1330), vascular tissue specific promoters (as mentioned in patent application US20030175783, Peleman et al., 1989 Gene 84; 359-369), specific stamen promoters (WO8910396, WO9213956), specific promoters of the dehiscence zone (WO9713865); and the like.

[53] The transcription termination signal and the polyadenylation region of the present invention includes, but is not limited to, SV40 termination signal, HSV TK adenylation signal, Agrobacterium tumefasciens nopaline synthase gene termination signal, signal termination of the CaMV RNA 35S gene, termination signal of the virus that attacks the subterranean Trifolium (SCSV), termination signal of the trpC gene of Aspergillus nidulans, and the like.

[54] The term expression cassette or DNA construct or expression construct refers to a nucleic acid construct that, when introduced into a

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18/36 host cell, results in the transcription and / or translation of an RNA or polypeptide, respectively. Sense and antisense constructions that are not or cannot be translated are expressly included in this definition. In the case of transgene overexpression and suppression of endogenous genes (for example, antisense, RNAi or sense suppression) a specialist will recognize that the inserted polynucleotide sequence does not need to be identical, but may only be substantially identical to a gene sequence from from which it was derived. As explained herein, these substantially identical variants are specifically covered by reference to a specific nucleic acid sequence. An example of an expression cassette is a polynucleotide construct that comprises a polynucleotide sequence that encodes the BAHD1 protein operably linked to a heterologous promoter. In some embodiments, an expression cassette comprises a polynucleotide sequence that encodes the BAHD1 protein that is directed to a position in the genome of a plant so that expression of the polynucleotide sequence is driven by a promoter that is present in the plant .

[55] "dsRNA" or "double-stranded RNA" refers to the structure formed between the sequence of the mRNA or sense RNA, the sequence of a spacer region and the sequence of the antisense RNA, with the "spacer region" referring to to the nucleotide sequence that is not related to the sequence of the target gene, such as the sequence of an intron.

[56] One way to produce dsRNA is by inserting into the DNA molecule a nucleotide sequence of the target gene in the sense orientation, and a nucleotide sequence in the antisense orientation, whether or not there is a spacer region between the sequences of sense and antisense nucleotides. The mentioned nucleotide sequences can consist of about 19nt to 2000nt or about 5000 nucleotides or more.

[57] Conveniently, dsRNA (double-stranded RNA) as described can be introduced into host cells by introducing and possibly integrating a gene construct containing the nucleic acid molecules of the present invention, transcribing them to produce dsRNA. Therefore, in another embodiment, the invention is also supported by having a genetic construction that is

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19/36 capable of being expressed in cells of eukaryotic organisms of interest, operationally linked to a DNA molecule that when transcribed produces a dsRNA molecule.

[58] The term "vector" refers to a replicon, such as a plasmid, phage or virus, to which other genetic sequences or elements (whether DNA or RN A) can be linked. In this way, the genes can be replicated together with the vector. Such vectors can be obtained commercially, including those supplied by Clontech Laboratories, Inc (Paio Alto, Calif.), Stratagene (La Jolla, Calif), Invitrogen (Carlsbad, Calif.), New England Biolabs (Beverly, Mass.) And Promega (Madison, Wis.). Some examples of vectors, but not limited, are the pKannibal vectors (Wesley SV, Heliwell CA, Smith NA, Wang M, Rouse DT, Liu Q, Gooding PS, Singh SP, Abbott D, Stoutjesdijk PA, Robinson SP, Gleave AP, Green AG, Waterhouse PM (2001) Construct design for efficient, effective and highthroughput Gene silencing in plants. Plant J. 27: 581590) pGLYP, pAC321, pCambia series (BioForge Co.), pBI121 (Chen, Ρο-Yen; Wang, Chen-Kuen; Soong, Shaw-Ching; To, Kin-Ying. Complete sequence of the binary vector pBI121 and its application in cloning T-DNA insertion from transgenic plants .. Molecular Breeding vol. 11 issue 4 May 2003. p. 287 -293), pBSK (Addgene Co.), pGEM-T easy (Promega Corporation), pETIOl / D-TOPO (Invitrogen). Obtaining recombinant vectors comprising promoters linked to nucleic acids is known in the art and can be found in Sambrook et al. (Sambrook, J., Russell, D.W., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press. 1989).

[59] The term "expression" refers to the transcription of a given DNA sequence. More specifically in that invention, it relates to the transcription and stable accumulation of double-stranded RNA derived from the nucleic acid fragments of the invention.

[60] "Interference inhibition" or "expression suppression" refers to the production of dsRNA transcripts capable of preventing expression of the target gene.

[61] "Transformation" refers to the transfer of the exogenous gene into the genome of a host organism and its consequent inclusion in the genetically stable inheritance.

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20/36 [62] “Plants” refer to multicellular, eukaryotic, autotrophic and photosynthetic organisms, belonging to the Plantae Kingdom. The term plant used here can refer to any plant or part of a plant, for example, seeds, and includes plants with varying levels of ploidy, including aneuploidy, polyploid, diploid and haploid. The term plant part, used here, refers to shoots, vegetative organs and / or structures (for example, leaves, stems and tubers), branches, roots, flowers and floral organs (for example, bracts, sepals, petals, stamens, carpels and anthers), eggs (including antipode cells, synergids and polar nuclei), seeds (including zygote, embryo, endosperm and seed coating), fruit (for example, the mature ovary), seedlings and plant tissues ( for example, vascular tissue, underground tissue and the like), as well as individual cells, groups of cells (for example, cultured plant cells), protoplasts, plant extracts and seeds. The class of plants that can be used in the methods of the invention is generally as wide as upper and lower vegetables subject to transformation techniques, including angiosperms (monocots and dicots), gymnosperms, pteridophytes, bryophytes and algae.

[63] The present invention also includes providing dsRNA molecules, which can be obtained by transcribing the molecules contained in the gene constructs, and which are useful for the methods according to the invention.

[64] Another object of the present invention is to provide eukaryotic cells and eukaryotic organisms containing dsRNA molecules of the invention, or containing the chimeric genes or gene constructs capable of producing dsRNA molecules of the invention. Gene constructs can be stably integrated into the cell genome of eukaryotic organisms.

[65] The gene constructs or chimeric genes of the invention can be introduced into the genome of the desired host plant through a variety of conventional techniques. For example, the construction can be introduced directly into the plant tissue using ballistic methods, such as bombarding particles covered with DNA, as described in Rech EL, Vianna GR, Aragão FJL (2007) High transformation efficiency by biolistics of soybean, bean and cotton

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21/36 transgenic plants, Nature Protocols, 3 (3): 410-418. They can also be introduced directly into the plant cell's genomic DNA using techniques such as electroporation and microinjection of plant cell protoplasts. Another way of introducing exogenous DNA into plants is using Agrobacterium tumefaciens, as described in Martins et al. A simple and highly efficient Agrobacterium-mediated transformation protocol for Setaria viridis. Biotechnology Reports, v. 6, p. 41-44, (2015).

[66] Transformed plant cells that are derived from any of the transformation techniques described above can be grown to regenerate an entire plant that has the transformed genotype and, consequently, the expected phenotype. Such regeneration techniques rely on the manipulation of certain phytohormones in tissue culture growth medium, typically containing a biocidal and / or herbicide marker, which must be introduced together with the desired nucleotide sequence. Plant regeneration from protoplast culture is described in Evans et al., Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMillilan Publishing Company, New York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985 (as mentioned in patent application US20020152501). Regeneration can also be achieved through plant calluses, explants, organs, or part of it. Such regeneration techniques are generally described in Klee et al., Ann. Ver. Of Plant Phys. 38: 467-486, 1987 1985 (as mentioned in the patent application

US20020152501).

[67] The term biomass, used here, refers to plant material that is processed to generate a product, for example, biofuel, such as ethanol, or animal feed, or cellulose for the paper and cellulose industry. Such plant material may include whole plants or parts of plants, for example, stems, leaves, branches, shoots, roots, tubers and the like.

[68] The term decrease in ferulic acid content in the context of this invention refers to a decrease in the amount of ferulic acid present in the cell wall or leaf in a genetically modified plant of the present invention, in

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22/36 compared to a wild type plant. In the present invention, the decrease in ferulic acid is normally considered to be decreased when the amount of ferulic acid in the cell or leaf wall is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70 %, 80%, 90% or more in relation to the amount of ferulic acid in the cell wall or leaf of a wild type plant. The ferulic acid content can be assessed using any method known in the art.

[69] The term decrease in ferulic acid content also encompasses embodiments where the ratio of ferulic acid to p-cumaric acid is reduced compared to a wild type plant. Thus, in some embodiments, the amount of ferulic acid can be the same as that of a wild-type plant, but in relation to p-cumárico, it can be increased, thus decreasing the ratio of ferulic acid to p-cumárico, when compared with a wild type plant. In the present invention, the ratio of ferulic acid to /? - cumharic is normally considered to be decreased when the ratio in the cell wall or leaf is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in relation to the ratio of the amount of ferulic acid in the cell wall or leaf in a wild type plant.

[70] The term saccharification reaction refers to a process of converting biomass, usually cellulosic or lignocellulosic biomass, into monomeric sugars, such as glucose and xylose.

[71] The term soluble sugar refers to a monomeric, dimeric or trimeric sugar that is produced from the saccharification of biomass.

[72] The term increased amount refers to an amount of sugar or soluble sugar obtained from a manipulated plant of the present invention, refers to an increase in the amount or yield of sugar that is obtained from the saccharification of biomass by amount of starting material, compared to the corresponding wild-type plant biomass. In the context of the present invention, corresponding wild-type plant biomass refers to plant material that is from the same part of the plant, such as biomass from a plant genetically modified for hydroxycinnamic acid levels. As understood in art, the

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23/36 increase in quantity or increase in yield is based on comparisons of the same amount of material from the corresponding plant.

[73] The term conversion reaction, as used here, refers to a reaction that converts biomass into a form of bioenergy. Examples of conversion reactions include, but are not limited to, combustion (burning), gasification, pyrolysis, and polysaccharide hydrolysis (enzymatic or chemical).

[74] The term increase in production, when referring to a quantity of bioenergy production obtained from a genetically modified plant of the present invention, refers to an increase in the amount of bioenergy that is produced from the biomass of a genetically modified plant subjected to a conversion reaction (for example, combustion, gasification, pyrolysis or polysaccharide hydrolysis), compared to the amount of bioenergy that is produced from the corresponding wild-type plant biomass.

[75] As used herein, the term BAHD1 acyl transferase refers to an acyl transferase that participates in the modification of a grass's arabinoxylan. The term encompasses variants and interspecies homologous to the specific polypeptides described herein. A nucleic acid encoding a BAHD1 acyl transferase refers to a gene, messenger pre-RNA, messenger RNA, and the like, including nucleic acids encoding polymorphic variants, alleles, mutants and homologs between species of the amino acid sequences described here . As used herein, the term BAHD3 acyl transferase refers to an acyl transferase that participates in the modification of a grass's arabinoxylan. The term encompasses variants and interspecies homologous to the specific polypeptides described herein. A nucleic acid encoding a BAHD3 acyl transferase refers to a gene, messenger pre-RNA, messenger RNA, and the like, including nucleic acids encoding polymorphic variants, alleles, mutants and homologs between species of the amino acid sequences described here . As used herein, the term BAHD5 acyl transferase refers to an acyl transferase that participates in the modification of a grass's arabinoxylan. The term encompasses variants and interspecies homologous to the specific polypeptides described herein. A nucleic acid encoding an acyl transferase of

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24/36

BAHD5 refers to a gene, pre-messenger RNA, messenger RNA, and the like, including nucleic acids encoding polymorphic variants, alleles, mutants and homologs between species of the amino acid sequences described herein. As used herein, the term BAHD8 acyl transferase refers to an acyl transferase that participates in the modification of a grass's arabinoxylan. The term encompasses variants and interspecies homologous to the specific polypeptides described herein. A nucleic acid encoding a BAHD8 acyl transferase refers to a gene, messenger pre-RNA, messenger RNA, and the like, including nucleic acids encoding polymorphic variants, alleles, mutants, and homologs between species of the amino acid sequences described here .

[76] The terms reduced activity level, reduced activity and decreased activity refer to a reduction in the amount or enzymatic activity of the BAHD1, BAHD3, BAHD5 and BAHD8 polypeptides by manipulating the BAHD1, BAHD3, BAHD5 and BAHD8 genes to decrease the amount of activity compared to non-GM. In some embodiments, reduced activity is a result of reduced levels of expression. A reduction in the level of activity or a reduced level of expression can be a reduction in the amount of activity or expression of the BAHD1, BAHD3, BAHD5 and BAHD8 genes of at least 10%, 20%, 30%, 40%, 50% , 60%, 70%, 80%, or 90% or greater. In some embodiments, the reduced level of activity or reduced level of expression occurs throughout all tissues of the transformed plant. In some embodiments, the reduction in the amount of activity or expression is located in one or more tissues of the transformed plant, such as in the cytosol. In some embodiments, BAHD1, BAHD3, BAHD5 and BAHD8 are not reduced in quantity, but are modified in the sequence of amino acids so that the enzymatic activity is reduced directly or indirectly. Reduction of the expression or activity of the BAHD1, BAHD3, BAHD5 and BAHD8 genes or polypeptides can be evaluated by any number of assays, including, but not limited to, the measurement of the level of RNA encoded by the BAHD1, BAHD3, BAHD5 and BAHD8 genes, the level polypeptides BAHD1, BAHD3, BAHD5 and BAHD8, the level of enzymatic activities

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25/36 of BAHD1, BAHD3, BAHD5 and BAHD8, or by measuring cell wall ferulic acid and / or cell wall p-cumaric acid content.

[77] The invention provides a method for generating plants with reduced levels of ferulic acid and increased saccharification. Transgenic plants produced by this strategy can be used both for the production of biofuel via second generation ethanol (2G) and for the production of animal feed.

[78] In one aspect, the invention provides a method of producing a plant with altered cell wall structure by inserting into said plant a DNA molecule whose expression results in an RNA molecule whose sequence comprises at least a segment of 18 or more contiguous nucleotides complementary to at least one fragment of SEQ ID No 1, SEQ ID No 3, SEQ ID No 5 or SEQ ID No 7. The said nucleotide segment can be transcribed in sense and antisense orientations, resulting in the RNA, by less in part, double tape. The invention also provides for the plant and / or parts of the plant produced by said method. Such plant may belong to the family Poaceae, more specifically the species Brachypodium distachyon, Setaria viridis, Setaria italica, Saccharum spp., Saccharum offlcinarum, Saccharum spontaneum, Zea mays, Sorghum bicolor, Brachiaria spp., Brachiaria brizantha, Brachiaria ruziziensis, Pennisetum spp., Panicum spp., Miscanthus spp., Erianthus spp., Triticum aestivum, Secale cereale, Hordeum vulgare, Avena spp., Avena sativa, Brachypodium spp., Setaria spp., Oryza sativa, Oryza spp. , Paspalum spp., Phalaris arundinacea, Miscanthus x giganteus, Lolium multiflorum, Lolium spp., Festuca spp., Arundo donax. Fruit, seed or propagable parts of said plant are also provided.

[79] In another aspect, the invention provides an isolated nucleic acid molecule comprising a nucleic acid sequence with at least 90, 95, 99 or 100% similarity to the sequence described in SEQ ID No 9, SEQ ID No 10, SEQ ID No 11 or SEQ ID No 12. An isolated nucleic acid molecule comprising a nucleic acid sequence with at least 90, 95, 99 or up to 100% similarity to the complement of the sequence described in SEQ ID No 9 , SEQ ID NO 10, SEQ ID NO 11 or SEQ ID NO 12.

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26/36 [80] A further aspect of the invention provides a chimeric gene that comprises at least one polynucleotide comprising a nucleic acid sequence with at least 90, 95, 99 or 100% similarity to the sequence described in SEQ ID No 9, SEQ ID NO 10, SEQ ID NO 11 or SEQ ID NO 12 operationally linked to an active promoter. It is further envisaged that the chimeric gene may comprise a sequence according to SEQ ID No 13 or in SEQ ID No 14. Also addressed is a gene construct comprising one or more of said chimeric gene.

[81] The invention further provides for a gene construct comprising a first region with a sequence of 18 or more consecutive nucleotides having at least 90, 95, 99 or up to 100% similarity with at least one sequence of 18 or more consecutive nucleotides from sense nucleotide sequence selected from SEQ ID No 9, SEQ ID No 10, SEQ ID No 11 and SEQ ID No 12 and a second region with a nucleotide sequence of 18 or more consecutive nucleotides having at least 90, 95, 99 or even 100% sequence similarity with the complement of 18 or more consecutive nucleotides from at least one nucleotide sequence selected from SEQ ID No 9, SEQ ID No 10, SEQ ID No 11 and SEQ ID No 12. The first and second regions of the aforementioned gene construct may be able to form a double-stranded RNA region, which may contain a spacer region, with such a spacer region ranging from 100 to 800 nucleotides and po to be an intron.

[82] In another aspect, a method is provided for producing a transgenic plant cell, plant, seed or plant part capable of expressing a dsRNA capable of suppressing the expression of the BAHD1, BAHD3, BAHD5 or BAHD8 gene, through transformation of a plant cell with a gene construct as described above.

[83] The invention further provides a vector comprising an isolated nucleic acid molecule comprising a nucleic acid sequence with at least 90, 95, 99 or 100% similarity to the sequence described in SEQ ID No 9, SEQ ID No 10 , SEQ ID NO 11 or SEQ ID NO 12, as well as the double-stranded RNA molecule produced from the expression of said vector.

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27/36 [84] Cell and transformed plant comprising an isolated nucleic acid molecule comprising a nucleic acid sequence with at least 90, 95, 99 or 100% similarity to the sequence described in SEQ ID NO 9, SEQ ID No 10, SEQ ID No 11 or SEQ ID No 12, as well as seed or propagating part of the plant. Said cell can be a eukaryotic cell or even, more specifically, a plant cell.

[85] In another aspect, a method of altering cell wall structure by exposing plant or part of the plant to the double-stranded RNA molecule whose sequence comprises at least a segment of 18 or more consecutive nucleotides of at least a sequence selected from SEQ ID No 9, SEQ ID No 10, SEQ ID No lie SEQ ID No 12. It is further predicted that the double-stranded RNA molecule can be chemically synthesized or produced by expression in a microorganism or by expression in a plant cell .

[86] A composition containing an isolated nucleic acid molecule comprising a nucleic acid sequence with at least 90, 95, 99 or 100% similarity to the sequence described in SEQ ID No 9, SEQ ID No 10, SEQ ID No. 11 or SEQ ID No. 12. Such composition may comprise a solid, liquid, powder, suspension, emulsion, spray, encapsulated, microbeaded, particulate, film, matrix or seed treatment. A method of producing a plant with altered cell wall structure is also provided by exposing the plant to said composition. Said plant is also provided.

Experiments performed and results obtained:

Genetic constructions:

[87] Silencing regions have been determined (SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 31, SEQ ID NO 32, SEQ ID NO 33 and SEQ ID NO 34) and cloned into binary vectors. The vectors (Figure 1) have the hpt selection marker on their TDNA, which confers resistance to the antibiotic hygromycin

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28/36 (transformation of S. viridis) and the bar gene, which confers resistance to the herbicide glufosinate ammonium (transformation of sugarcane). In the vector for S. viridis, the expression of the hpt gene is driven by the 2x CaMV 35S constitutive promoter (vector for silencing BAHD1) and the constitutive promoter Actin in rice (Act-1) (vectors for silencing the BAHD3, BAHD5 and BAHD8 genes and vectors containing the secondary cell wall specific promoters SzIRX1 and /? z / IRX5). In the sugarcane vector, the expression of the selection marker gene (bar) is directed by the constitutive promoter Actin of rice (Act-1). In addition, the silencing cassettes in both vectors are directed by the promoters: corn ubiquitin (ZwUbi-1) for constitutive expression of the dsRNA and by the promoters SiIRX1 and Z? Z / 1RX5 for specific expression of the dsRNA in the secondary cell wall.

Genetic transformation and obtaining homozygous strains of Setaria viridis, Brachypodium distachyon and sugar cane:

[88] The genetic transformation of S. viridis was mediated ροτ Agrobacterium according to the protocol described by Martins et al., A simple and highly efficient Agrobacterium-mediated transformation protocol for Setaria viridis. Biotechnology Reports, v. 6, p. 41-44,2015. Embryogenic calluses obtained from mature S. viridis seeds were induced and used as an explant. Approximately 50 calli were incubated for 5 minutes in the Agrobacterium suspension containing the vectors described above. After 4 to 6 weeks in culture medium, the seedlings that survived the hygromycin selection were transplanted into pots and acclimated in Fitotron chambers. Molecular confirmation of the transgenia was performed by PCR with specific primers for the hpt gene (SEQ ID No 35 and SEQ ID No 36). For each construction, the transformation efficiency was calculated using the following formula: number of PCR positive plants / total number of inoculated calli x 100 (Table 1).

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Table 1 Genetic transformation of S. viridis embryogenic calli with BAHDs genes.

Gene District Attorney Number of embryogenic calluses Selection Marker Number of PCR positive plants Transformation Efficiency (%) ^a BAHDlri ZmUBI 50 hpt 13 26 BAHD5ri ZmUBI 50 hpt 5 10 BAHD8ri ZmUBI 50 hpt 5 10 BAHDlri SilRXl 50 hpt 7 14 BAHD5ri SilRXl 50 hpt 10 20 Efficiency transformation calculated by number of PCR plants

positive / total number of inoculated calli x 100.

Table 2 Example of segregation for hygromycin resistance in the Tl generation of plants transformed with the BAHD genes.

Event Generation Resistant ¹ Sensitive ^b Dex value ² to 3: l ^c Aspect ratio 3: 1 (P = 0.05) ^d UBlri-18 You 62 28 1.79 s UBlri-17 You 72 32 1.84 s UBlri -3 You 48 46 28.72 N UB5ri-4 You 83 17 3.41 s UB5ri -6 You 58 39 11.96 N UB8ri -4 You 30 8 0.31 s SBlri -2 You 31 14 0.86 s SB5ri -5 You 34 19 3.32 s

^a Number of surviving seedlings in the medium containing 30 mg L ' ¹ of hygromycin. ^b Number of sensitive seedlings in the medium containing 30 mg L ' ¹ of hygromycin. ^c Significantly different P <0.05.

^d Ratio of resistant vs sensitive seedlings in hygromycin [89] To obtain homozygous strains of positive events, segregation analysis in Tl seeds was performed to estimate the number of insertions, for each gene, in each event obtained. First, mature seeds from each event were treated with concentrated sulfuric acid, promoting dormancy break. At

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30/36 seeds were grown in selective culture medium containing 50 mg / L of hygromycin. After 7 days, the number of resistant and sensitive plants was evaluated and the proportion was statistically analyzed using the χ ² test (Table 2). Events with a single insertion segregating in the Mendelian proportion 3: 1 were selected (Ev.17.3 and Ev.18.1). The T2 seeds of these events were grown in a selective medium to select homozygous transgenic strains.

[90] The genetic transformation of B. distachyon was following the protocol described by Vogei J and Hill T. High-efficiency Agrobacterium-mediated transformation of Brachypodium distachyon inbred line Bd21-3. Plant Cell Reports, v. 27, n. 3, p. 471478,2008. Embryogenic calluses of the Bd21-3 strain were obtained from immature embryos and used as explants for genetic transformation via Agrobacterium tumefaciens.

[91] The sugarcane genetic transformation was mediated by Agrobacterium using embryogenic callus cultures derived from palm hearts aged 6-8 months of the commercial variety SP80-3280.

Relative quantification of BAHD gene expression in homozygous events of

S. viridis:

[92] The relative expression of the BAHD1, BAHD3, BAHD5 and BAHD8 genes in homozygous T0 and T2 transgenic plants was quantified using the real-time PCR technique as described by Martins, PK. et al. Selection of reliable reference genes for RTqPCR analysis during developmental stages and abiotic stress in Setaria viridis. Scientific Reports, v. 6, 2016. After the extraction of total RNA, cDNA synthesis and real-time PCR reaction, the quantification of gene expression in T0 and T2 plants, in relation to untransformed plants (NT), showed a significant reduction in the expression of genes in all tissues analyzed and constitutively under the control of the ZmUbi-1 promoter (Figure 2 and Figure 3, respectively). The same result was observed in plants whose genes were specifically silenced, under the control of the secondary cell wall promoter SzIRXl (Figure 4).

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Determination of phenolic acids (FA and pCA) bound to the cell wall of homozygous events of S. viridis:

[93] Phenolic acids bound to the cell wall of S. viridis samples (NT, Ev 17.3 and Ev 18.1) were quantified by ultra high performance liquid chromatography with a diode array detector (UHPLC-DAD). The preparation of the samples was according to the alkaline hydrolysis method (2M NaOH) of the alcohol insoluble residue (AIR) described by Molinari, HBC. et al., Grass cell wall feruloylation: distribution of bound ferulate and candidate gene expression in Brachypodium distachyon. Frontiers in Plant Science, v. 4, p. 50, 2013.

[94] Events Ev 17.3 and 18.1, with reduced levels of BAHD1 gene transcripts, should also produce less amount of ferulates linked to the cell wall and, thus, generate a reduction in cross-linking between its polymers. After quantification, the results showed a significant reduction in the content of trans-ferulic acid (TFA), in all tissues, when compared with the NT control (Figures 5, 6 and 7). However, in stem and root the content of / 2-cumárico increased significantly in relation to the NT (Figures 5 and 7).

Quantification of lignin by the method of acetyl bromide and lignin monomers on HPLC of homozygous events of S. viridis'.

[95] For the quantification of lignin, the samples were first dried, crushed and the biomass was thoroughly washed to remove interferents such as soluble proteins, phenolic compounds and hydrophobic proteins. Thus, 20 mg of the protein-free cell wall fraction was incubated with 0.5 mL of 25% (v / v) acetyl bromide at 70 ° C for 30 min. For the solubilization of lignin, 4 ml of cold acetic acid was used for leaf and stem samples and 5 ml for root samples. The samples were then centrifuged (3300 RPM for 5 min) and the diluted supernatant was used for the spectrophotometer readings (280 nm). The lignin concentration was finally determined according to a standard curve and the results

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32/36 expressed in mg lignin g ' ¹ of protein-free cell wall (Adapted from Moreira-Vilar, FC, et al. The acetyl bromide method is faster, simpler and presents best recovery of lignin in different herbaceous tissues than klason and thioglycolic acid methods. PloS one, v. 9, n. 10, p. el 10000,2014).

[96] As shown in figure 11A, the lignin content in the stem of transgenic events has not been changed. However, a small reduction was observed at the root (Figure 8C) of the two events in relation to the NT and an increase in the lignin content in the leaves of the Ev. 17.3 (Figure 8B).

[97] For the quantification of monomers by HPLC, 50 mg of the protein-free cell wall was used. The samples were incubated with 1 ml of 2M NaOH and 0.1 ml of nitrobenzene in an oven at 170 ° C for 150 minutes. After cooling, the samples were placed in a separating funnel for washing with distilled water. The phases were separated with chloroform and after discarding the chloroform phase the samples were acidified with HCL and a new phase separation was performed to collect the chloroform phase in a glass flask. The samples were rotoevaporated and resuspended in 1 mL of methanol PA, diluted 20 times in a mobile phase (20% methanol in 4% acetic acid) and read on HPLC, Cl8 column 15 cm, at a wavelength of 290 nm with a flow of 1 , 2 ml / min. (Adapted from Lima, RB. Et al. Enhanced lignin monomer production caused by cinnamic acid and its hydroxylated derivatives inhibits soybean root growth. PloS one, v. 8, n. 12, p. E80542, 2013).

[98] The results showed that there was no change in the monomers H, S and G in the culm of the events in relation to the NT (Figure 9). In the leaves and roots, there is an increase in monomers H and S in both events (Figure 9).

Enzymatic saccharification of homozygous events of 5. viridis'.

[99] Samples from events 17.3 and 18.1 and NT were subjected to saccharification (without pre-treatment) with commercial enzymatic cocktail Cellic®Ctec3 at 10 FPU (Filter paper units) / g of biomass. Each part of the plant (leaves and stem) was analyzed separately. Enzymatic hydrolysis was performed with 2% (w / v) of biomass in 0.5

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M citrate buffer pH 5.0 at 50 ° C with 1000 rpm agitation. Rates of 0, 6 and 24 hours were analyzed. The reactions were performed in 2 ml Eppendorf Thermomixer tubes using a microplate incubator (Eppendorf, Germany). At the end, the samples were centrifuged at 10.000g for 15 min (Centrifuge 5418, Eppendorf), and heated at 80 ° C for 5 minutes to inactivate the enzymes. Structural carbohydrates were determined according to the Laboratory Analytical Procedure Determination of Structural Carbohydrates and Lignin in Biomass, from the National Renewable Energy Laboratory (NREL). All dosages were performed in three biological replicates and each replica was dosed in triplicate. The results were expressed as averages in grams per liter. The statistical test adopted was bidirectional ANOVA followed by the Bonferroni test, with p <0.05 (*) and p <0.01 (**).

[100] The results showed that the most effective event to contribute to biomass hydrolysis was the Ev event. 18.1 in both the stem and the leaf (Figure 10). However, the culms were the samples that released more reducing sugar after 6 hours of saccharification (Table 3).

Table 3 Increase in the concentration of glucose released (in percentage) in the saccharification test for the two events of S. viridis in comparison with the control (Non-transgenic plant).

Samples Increased concentration of glucose released (%) Sheets Thatch 17ν 17.3-T6 12.97 45.29 18ν 18.1-T6 49.93 79.92 17ν 17.3-T24 15.29 37.17 18ν 18.1-T24 45.91 64.55

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Phenotyping of homozygous events of 5. viridis and sugarcane:

[101] For biomass sampling, 10 plants from each event and senescent NT were selected. The plant organs (excluding root) were completely dried in an oven at 65 ° C for 72 h to obtain the dry biomass of the aerial part. The seeds were collected after 90 days and the weight of a thousand seeds was estimated.

[102] Some studies suggest that TF A dimerization plays a role in plant growth by inhibiting cell wall elongation and expansion (Macadam, JW; Grabber, JH. Relationship of growth cessation with the formation of diferulate cross-links and p-coumaroylated lignins in tall fescue leaf blades .Plant, v. 215, n. 5, p. 785-793, 2002; Obel N et al., Dynamic changes in cell wall polysaccharides during wheat seedling development. Phytochemistry, v. 60, n. 6, pp. 603-610, 2002; Sasayama D et al., Involvement of cell wall-bound phenolic acids in decrease in cell wall susceptibility to expansins during the cessation of rapid growth in intemodes of floating rice. Physiology, v. 168, p. 121-127, 2011). Thus, with the reduction of the FA dimer content, smaller plants would be expected to decrease the ability to sustain themselves or larger ones, due to the greater expansion of cells during growth. In fact, corn progenies with reduced ferulic acid esters in the seedling phase, had an increase of approximately 8% of the biomass in field trials (Jung, HG & Phillips, RL. Putative seedling ferulate ester maize mutant: morphology, biomass yield , and stover cell wall composition and rumen degradability (Crop Science, v. 50, η. 1, p. 403-418, 2010). In transgenic plants of 5. viridis we observed a significant increase in biomass and in the number of seeds of Ev. 18.1 in relation to the NT. However, for the EV. 17.3 there was no significant difference in biomass compared to NT (Figure 11).

[103] Regarding the transgenic sugarcane plants in Figure 14D, it is possible to observe that there is no phenotypic difference between the untransformed plant (NT) and the transgenic events.

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Relative quantification of BAHD1 gene expression in sugarcane events:

[104] The relative expression of the BAHD1 gene in transgenic plants was quantified using the real-time PCR technique as described by Reis RR. et al., Induced over-expression of AtDREB2A CA improves drought tolerance in sugarcane. Plant Science, Vol. 221-222, p. 59-68, 2014. After extracting the total RNA from the leaves, cDNA synthesis and real-time PCR reaction, the quantification of the expression of the BAHD1 gene in the 14 events generated, in relation to the untransformed plant (NT), showed a reduction expression of BAHD1 in 7 events (Figure 12).

Determination of phenolic acids (FA and pCA) linked to the cell wall of sugarcane events:

[105] Phenolic acids bound to the cell wall of sugarcane samples (NT, Ev. 1.0, Ev. 2.4, Ev. 3.6, Ev. 4.0 and Ev. 5.0) were quantified by ultra high-performance liquid chromatography performance with diode array detector (UHPLC-DAD). The preparation of the samples was in accordance with the alkaline hydrolysis method (2M NaOH) of the alcohol insoluble residue (AIR) described by Molinari HBC et al., Grass cell wall feruloylation: distribution of bound ferulate and candidate gene expression in Brachypodium distachyon. Frontiers in Plant Science, v. 4, p. 50, 2013.

[106] Events with reduced levels of BAHD1 gene transcripts should also produce lesser amounts of ferulates bound to the cell wall and, thus, generate a reduction in cross-linking between its polymers. After quantification, the results showed a significant reduction in the content of trans-ferulic acid (TFA), in the leaves, of the events Ev 1.0, Ev 3.6 and Ev. 4.0, when compared to the NT control (Figure 13A). In the case of Ev 2.3, the reduction was so drastic that the amount of TFA cannot be detected because it is below the lowest point on the calibration curve of the method. The content of / j-cumárico (pCA) increased significantly in relation to NT only in Ev 2.4 and decreased in Ev 2.3 (Figure 13B).

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Enzymatic saccharification of sugarcane events:

[107] Leaf samples from events 2.3, 3.1 and 4.0 and NT were subjected to saccharification (without pre-treatment) with commercial enzymatic cocktail Cellic®Ctec 3 to 5 FPU (Filter paper units) / g of biomass. Enzymatic hydrolysis was carried out with 2% (w / v) biomass in 0.5 M citrate buffer pH 5.0 at 50 ° C with 200 rpm agitation. Aliquots were removed after 0, 3, 6, 10 and 24 hours of enzymatic hydrolysis and placed in 2 ml Eppendorf. The samples were centrifuged at 10.000g for 15 minutes (Centrifuge 5418, Eppendorf) and the supernatant stored for carbohydrate analysis. Structural carbohydrates were determined according to the Laboratory Analytical Procedure Determination of Structural Carbohydrates and Lignin in Biomass, from the National Renewable Energy Laboratory (NREL). All dosages were performed in three biological replicates and each replica was dosed in triplicate. The results were expressed as averages in grams per liter. The statistical test adopted was bidirectional ANOVA followed by the Bonferroni test, with p <0.05 (*) and p <0.01 (**).

[108] The results showed that the most effective event to contribute to biomass hydrolysis was the Ev event. 2.3 at 6, 10 and 24 hours of hydrolysis (Figure 15). But the three events showed an increase in saccharification at times 6, 10 and 24 hours compared to the control.

Claims (39)

CLAIMS: 1. Method of producing a plant with an altered cell wall structure characterized by inserting into said plant a DNA molecule whose expression results in an RNA molecule in which the sequence comprises at least a segment of 18 or more contiguous nucleotides complementary to at least a sequence fragment selected from SEQ ID No 1, SEQ ID No 3, SEQ ID No 5 and SEQ ID No 7. Method according to claim 1 characterized in that said nucleotide segment is transcribed in sense and antisense orientations, resulting in the RNA, at least in part, double stranded. Plant characterized in that it is produced by the method of claim 1. 4. The plant of claim 3, characterized by belonging to the Poaceae family. 5. The plant of claim 3 characterized by being selected from the species Brachypodium distachyon, Setaria viridis, Setaria italica, Saccharum spp., Saccharum offlcinarum, Saccharum spontaneum, Zea mays, Sorghum bicolor, Brachiaria spp., Brachiaria brizantha, Brachiaria ruzizi , Pennisetum spp., Panicum spp., Miscanthus spp., Erianthus spp., Triticum aestivum, Secale cereale, Hordeum vulgare, Avena spp., Avena sativa, Brachypodium spp., Setaria spp., Oryza sativa, Oryza spp. ., Paspalum spp., Phalaris arundinacea, Miscanthus x giganteus, Lolium multiflorum, Lolium spp., Festuca spp., Arundo donax. 6. Fruit, seed or propagable parts characterized by the plant of claim 3. 7. Isolated nucleic acid molecule characterized by comprising a nucleic acid sequence with at least 90% similarity to a sequence selected from SEQ ID No 9, SEQ ID No 10, SEQ ID No 11 and SEQ ID No 12. Nucleic acid molecule according to claim 7, characterized in that it has at least 95% similarity with a sequence selected from SEQ ID No 9, SEQ ID No 10, SEQ ID No 11 and SEQ ID No 12. Petition 870180124196, of 08/31/2018, p. 5/10 2/6 Nucleic acid molecule according to claim 8, characterized in that it has at least 99% similarity with a sequence selected from SEQ ID No 9, SEQ ID No 10, SEQ ID No 11 and SEQ ID No 12. Nucleic acid molecule according to claim 9, characterized in that it comprises a nucleic acid sequence selected from SEQ ID No 9, SEQ ID No 10, SEQ ID No 11 and SEQ ID No 12. 11. Isolated nucleic acid molecule characterized by comprising a nucleic acid sequence with at least 90% similarity to the complement of a sequence selected from SEQ ID No 9, SEQ ID No 10, SEQ ID No 11 and SEQ ID No 12. Nucleic acid molecule according to claim 11, characterized in that it has at least 95% similarity with the complement of a sequence selected from SEQ ID No 9, SEQ ID No 10, SEQ ID No 11 and SEQ ID No 12. Nucleic acid molecule according to claim 12, characterized in that it has at least 99% similarity with the complement of a sequence selected from SEQ ID No 9, SEQ ID No 10, SEQ ID No 11 and SEQ ID No 12. Nucleic acid molecule according to claim 13, characterized in that it comprises a nucleic acid sequence complementary to a sequence selected from SEQ ID No 9, SEQ ID No 10, SEQ ID No 11 and SEQ ID No 12. 15. Chimeric gene characterized by comprising: i. at least one polynucleotide defined in any one of claims 7 to 14; and ii. an active promoter, operationally linked to the polynucleotide defined in (i). Petition 870180124196, of 08/31/2018, p. 6/10 3/6 Chimeric gene according to claim 15, characterized in that it comprises a sequence according to the sequence described in SEQ ID No 13 or in SEQ ID No 14. 17. Gene construction characterized by comprising one or more chimeric genes defined in claim 15. 18. Gene construction characterized by understanding: The. a first region with a sequence of 18 or more consecutive nucleotides having at least 90% similarity to at least one sequence of 18 or more consecutive nucleotides of the sense nucleotide sequence selected from SEQ ID No 9, SEQ ID No 10, SEQ ID No 11 and SEQ ID NO 12; B. a second region with a nucleotide sequence of 18 or more consecutive nucleotides having at least 90% sequence similarity to the complement of 18 or more consecutive nucleotides of at least one nucleotide sequence selected from SEQ ID No 9, SEQ ID No 10 , SEQ ID No 11 and SEQ ID No 12. 19. Gene construction according to claim 18, characterized by comprising: The. a first region with a sequence of 18 or more consecutive nucleotides having at least 95% similarity to at least one sequence of 18 or more consecutive nucleotides of the sense nucleotide sequence selected from SEQ ID No 9, SEQ ID No 10, SEQ ID No 11 and SEQ ID NO 12; B. a second region with a nucleotide sequence of 18 or more consecutive nucleotides having at least 95% sequence similarity to the complement of 18 or more consecutive nucleotides of at least one nucleotide sequence selected from SEQ ID No 9, SEQ ID No 10, SEQ ID NO 11 and SEQ ID NO 12. 20. Gene construction according to claim 19 characterized by comprising: The. a first region with a sequence of 18 or more consecutive nucleotides having at least 99% similarity to at least Petition 870180124196, of 08/31/2018, p. 7/10 4/6 a sequence of 18 or more consecutive nucleotides from the sense nucleotide sequence selected from SEQ ID No 9, SEQ ID No 10, SEQ ID No 11 and SEQ ID No 12; B. a second region with a nucleotide sequence of 18 or more consecutive nucleotides having at least 99% sequence similarity to the complement of 18 or more consecutive nucleotides of at least one nucleotide sequence selected from SEQ ID No 9, SEQ ID No 10 , SEQ ID No 11 and SEQ ID No 12. 21. Gene construction according to claim 20 characterized by comprising: The. a first region with a sequence of 18 or more consecutive nucleotides from at least one sequence selected from SEQ ID No 9, SEQ ID No 10, SEQ ID No 11 and SEQ ID No 12; B. a second region with a nucleotide sequence of 18 or more consecutive nucleotides complementing 18 or more consecutive nucleotides of the nucleotide sequence of at least one sequence selected from SEQ ID No 9, SEQ ID No 10, SEQ ID No 11 and SEQ ID No 12. 22. Gene construction according to any one of claims 17 to 21, characterized in that the first and second regions are capable of forming a double-stranded RNA region, which may, in addition to the total length of the first and second regions, a spacer region. 23. Gene construction according to claim 22 characterized in that the spacer region between the first and the second region ranges from 100 to 800 nucleotides. 24. Gene construction according to claim 22 or 23, characterized in that the spacer sequence is an intron. 25. Method of producing a transgenic plant cell, plant, seed or plant part capable of expressing a double-stranded RNA capable of suppressing the expression of at least one gene selected from BAHD1, BAHD3, BAHD5 and BAHD8, characterized by transforming a plant cell with a gene construct defined in any one of claims 17 to 24. Petition 870180124196, of 08/31/2018, p. 8/10 5/6 26. Vector characterized in that it comprises the isolated nucleic acid molecule according to any one of claims 7 to 14. 27. Double-stranded RNA molecule characterized by being produced from the expression of a nucleic acid molecule according to claim 26. 28. Transformed cell characterized by comprising at least one nucleic acid molecule according to any one of claims 7 to 14. 29. A cell according to claim 28 characterized in that it is a eukaryotic cell. Cell according to claim 29, characterized in that it is a plant cell. 31. Transformed plant characterized by comprising at least one nucleic acid molecule according to any one of claims 7 to 14. 32. Transformed plant characterized by comprising the nucleic acid molecule defined in claim 27. 33. Transgenic seed or propagating part characterized by the plant of claim 31 or 32. 34. Method of altering cell wall structure characterized by exposing plant or part of the plant to the double-stranded RNA molecule whose sequence comprises at least one segment of 18 or more consecutive nucleotides from at least one sequence selected from SEQ ID No 9, SEQ ID NO 10, SEQ ID NO 11 and SEQ ID NO 12. 35. The method of claim 34 characterized in that the double-stranded RNA molecule is chemically synthesized or produced by expression in a microorganism or by expression in a plant cell. 36. Composition characterized by containing a molecule according to any of claims 7 to 14. 37. The composition of claim 36 characterized in that the composition comprises a solid, liquid, powder, suspension, emulsion, spray treatment, Petition 870180124196, of 08/31/2018, p. 9/10 6/6 encapsulated, with microbeads, with particulate carriers, in film, in matrix or seed. 38. Method of producing a plant with altered cell wall structure characterized by placing the plant in contact with the composition defined in claim 37. 39. Plant with altered cell wall structure characterized by having been treated with the composition defined in claim 37.