WO2003018819A1

WO2003018819A1 - Method for obtaining transformed corn plants with improved properties of digestibility, resulting corn plants and uses

Info

Publication number: WO2003018819A1
Application number: PCT/FR2002/002982
Authority: WO
Inventors: Magalie Pichon; Michel Beckert; Isabelle Nadaud; Deborah Goffner
Original assignee: Genoplante-Valor
Priority date: 2001-08-31
Filing date: 2002-08-30
Publication date: 2003-03-06
Also published as: EP1425401A1; FR2829151A1; CA2459076A1

Abstract

The invention concerns the use of a polynucleotide modulating the synthesis of COMT methyltransferase of corn encoded by the nucleotide sequence SEQ ID No. 1, in a method for obtaining corn plants with improved properties of digestibility having a modified ratio between the S and G units of lignin and having a modified lignin content which remains compatible with the normal development of the plant. The invention is useful for improving digestibility of corn.

Description

Process for obtaining transformed corn plants with improved digestibility characteristics, corn plants obtained by the process and uses

The present invention relates to the field of improving the digestibility characteristics of corn, by modifying the composition and content of lignins.

PRIOR ART Corn silage is an interesting food from several points of view. For the farmer, the yield in the field of corn is relatively high, harvesting and storage are easy. For animals, in particular for dairy cows, the nutritional qualities of ensiled corn are stable and can easily be supplemented with proteins by other forage silage or by soybean meal. These nutritional qualities allow the persistence of milk production. Thus, a mixture of corn silage and fodder silage makes it possible to enhance the use of fodder proteins whose solubility (50%) is very high and very fast at the level of the cow's rumen (Léonard, 1996) . The low calcium and potassium content of corn silage makes it possible to dilute the important content of these elements in forage silage, in particular those of legume silage and to obtain an almost perfect balance between energy and protein from start to finish of lactation. Silage is often an element that helps the breeder to control energy-protein synchronism. Given the nutritional qualities of corn silage, the cultivation of this type of fodder is widespread and covers more than 3 million hectares in the European Union. Optimizing the qualities of corn silage consists in increasing the net energy provided by this type of food by improving its digestibility.

An important factor in reducing the digestibility of fodder corn is linked to the presence in the walls of plant cells of phenolic compounds, in particular lignins. Lignins establish different types of link with the other parietal constituents to form a tight mesh which hinders the accessibility of carbohydrates with digestive enzymes, the main source of energy for herbivores. Depending on their stage of maturity, plants would not have the same type of lignins deposited on their cell walls. The increase in lignification during the maturation of the plant causes a decrease in the digestibility of this plant (Bentley, 1959 and Grabber et al., 1992). It is difficult to correlate the reduction in the digestibility of plants during their maturation with lignin content, lignin composition, lignin structure, or with any other parietal component given the changes in chemical constituents and structure physical associated with the maturation of the plant.

Consequently, one of the preferred ways of improving the qualities of corn silage concerns the modification of the composition and lignin content of fodder plants. Indeed, a slight decrease in the lignin content leads to a strong improvement in digestibility. An experiment conducted by Emile (1995) shows that a more digestible variety makes it possible to increase the level of milk production by more than one kilogram and the weight gain of cows by 22 kg compared to a less digestible variety. Thus, the more digestible the variety, the higher the milk production and the less the loss of flesh state.

Brown midrib corn, in particular bm3 corn, has a greatly increased digestibility compared to conventional corn and significantly improves milk production. These bm3 maize differ from “normal” maize in a greatly reduced lignin content (up to 40%) and modified S (sinapyl alcohol) / G units (coniferyl alcohol) unit ratios. However, the drawbacks of bm3 maize such as a reduced field yield, an increased susceptibility to lodging, a lack of growth and vigor at the start of vegetation and a delay in flowering prevent their exploitation (Barrière and Argillier ( 1993)).

Lignin is a major compound in the cell wall of sclerenchyma and the xylem of vascular plants. It plays an important role in the conductive functions of xylem by reducing the water permeability of cell walls. In lignified tissues, it acts as an intercellular liaison officer. It is responsible for the rigidity of cell walls and the upright growth of plants and participates in the resistance of plants to mechanical attack (wind, injury, etc.) or infection by pathogens, tolerance to thermal and water stress.

In general, it is considered that lignin is an insoluble polymer of 3 monomers of alcohols or monolignols: p-coumaryl alcohol (units H), coniferyl alcohol (units G) and sinapylic alcohol (units S) . Each type of precursor can form a variety of bonds with other precursors and thus constitute lignin. Other links can also be established with other wall compounds (polysaccharides and proteins) to form a complex three-dimensional network. The content and composition of lignins vary between the major groups of higher plants and between species. The two major classes are gymnosperm lignins which mainly contain G units and a small proportion of H units, and angiosperm lignins which contain both S, G units and only a small proportion of H units (Whetten and al., 1998). Monocotyledon lignins, and more particularly cereals, are the most complex. They consist of the 3 units H, G, S to which are added in a significant proportion of hydroxycinnamic acids (ferulic and coumaric acid) linked by ethers or esters bonds. This structural heterogeneity is also found within the same plant, depending on the organ considered but also according to the stage of development and the location in the wall (Lewis and Yamamoto, 1990)).

The lignin biosynthesis pathway is shown in Figure 1. Although the lignin biosynthesis pathway is much studied, many steps are still not understood, especially those involved in methoxylation.

The current hypothesis of the monolignol biosynthetic pathway considers that the metabolic network leading to the formation of the S and G units involves successive reactions of hydroxylation and O-methylation. The phenylpropanoides biosynthesis pathway begins with the conversion of phenylalanine to cinnamic acid under the action Phenylalanine-Ammonia-Lyase (PAL). The product of PAL activity on phenylalanine is trans-cinnamic acid which is a substrate for cinnamate-4-hydroxylase (C4H), a P450-hydroxylase. C4H hydroxylates the 4 position of cinnamate to produce p-coumaric acid. In monocots, p-coumaric acid results from the action of Tyrosine-Ammonia-Lyase (TAL) on tyrosine.

All of the enzymes subsequently involved in the metabolic network have been the subject of reviews (Boudet et al., 1995; Dixon et al., 2001; Whetten et al., 1998, Li et al., 2000), they include : - O-methyltransferases having different designations according to the species: caffeic acid 3-O-methyltransferase (COMT) also designated caffeic acid O-methyltransferase (COMT), O-methyltransferase COMT or even preferentially in the text methyltransferase COMT or 5-hydroxyconiferyl aldehyde O-methyltransferase (AldOMT) and the

Caféoyl coenzyme A 3-O-methyltransferase (CCOAMT), hydroxycinnamate coenzyme A ligases (4CL), a ferulate 5-hydroxylase (F5H) with cytochrome P450, p-coumarate hydroxylase (C3H), - and several isoforms of Cinnamoyl Co A reductase (CCR) and

Cinnamyl alcohol dehydrogenase (CAD). The final stage of polymerization of the monolignols probably involves parietal enzymes, peroxidases and / or laccases. The enzymes catalyzing the methylation steps are O-methyltransferases. O-methyltransferases (S-adenoxyl-L-methionine O-methyltransferases, EC 2.1.1.6) play an important role in the biosynthesis of monolignols. It is currently accepted that COMT would preferentially intervene in the synthesis of S units and CCoAOMT in the synthesis of G units.

COMT would allow the methylation of the free forms of hydroxycinnamic acids: caffeic acid and 5-hydroxyferulic acid by introducing one or two methoxy groups respectively into the lignin monomers (Davin and Lewis, 1992). Recently, it has been shown in poplar that COMT is in fact a 5-hydroxyconiferyl aldehyde O-methyltransferase (AldOMT). In fact, 5-hydroxyconiferaldehyde is the preferred substrate for AldOMT. Thus, for Li et al. (2000), there are in woody angiosperms, two distinct hydroxylation / methylation pathways leading respectively to units S and G: CCoA3H (Coumaroyl CoA 3-hydroxylase) / CCoAOMT, with 4CL, CCR and CAD are associated to the synthesis of G units, and CAIdδH (Coniferaldehyde 5-hydroxylase) / AldOMT connects from conyferyl aldehyde for the biosynthesis of S units. The involvement of COMT in the biosynthesis of lignin S units is supported by strong reduction in lignin in S units but not of G units in transgenic plants under-expressing COMT such as tobacco (Atanassova et al., 1995), alfalfa (Guo et al., 2001) and poplar (Van Doorselaere and al., 1995).

The S and G units are therefore important components of lignins and therefore play a role in the digestibility of the wall of plant cells. However, the impact on digestibility of the increase or decrease in S and / or G units varies according to the studies. Thus, studies on the mutant bm3 have shown that the increased digestibility of the walls is associated with a strong alteration in the structure of lignins: low S / G ratio with substantial incorporation of 5-hydroxyguaiacyl (5-OH G) units (Lapierre et al., 1988) and a lower lignin content. If the correlation results between the digestibility and the lignin content of the plant are always negative, the correlation results between the S / G ratio and the lignin content and the digestibility are not always identical. Thus, for Zhong et al. (1998) and Méchin et al. (2000), the S / G ratio is positively correlated with digestibility. In other words, the respective results of these authors on tobacco and corn show that the more the S / G ratio increases, the more digestibility is important. Thus, depending on the species and the studies, it appears that a variation in the S / G ratio, associated or not with a reduced lignin content, may be accompanied by stability, a reduction or an increase in the digestibility.

These contradictory results demonstrate that it is not obvious to predict the consequences of the modification of the activity of O-methyltransferases (COMT, AldOMT) on the S / G ratio and the content of lignin and therefore on the digestibility of the plant. The invention described in this patent makes it possible to obtain corn plants offering better digestibility by modifying the O-methyltransferase activities.

In particular, a person skilled in the art is not able to transpose the highly contradictory experimental results described in the state of the art relating to the consequences of over-expression or, on the contrary, of under-expression of an enzyme involved in the biosynthesis of lignin in a given plant species, including maize, on the content and composition of lignin, in particular on the ratio between S units and G units.

In addition, the person skilled in the art, in the light of the experimental results described in the state of the art, could not have foreseen, in the specific case of corn, whether an increase or a reduction in the ratio S units / G units Lignin and / or the lignin content would have a positive or negative influence on the digestibility characteristics of this plant species or of the corresponding transformation products, for example the fodder produced from maize.

SUMMARY OF THE INVENTION It has been shown according to the invention that, surprisingly, the induction of the modulation of the synthesis of an enzyme involved in the biosynthesis pathway of corn lignin, caffeic acid 3- 0 Methyltransferase, also known as COMT, called in the remainder of this text methyltransferase COMT, caused a modification of the ratio S units / G units in the composition of lignin and / or the lignin content, compared to what is found in wild corn, and that the modification of the S units / G units ratio and / or of the lignin content gave the corn plant, as well as its transformation products, improved digestibility characteristics. By “modulation” of the synthesis of an enzyme is meant according to the invention a decrease, or on the contrary an increase in the synthesis of said enzyme.

By “modification” of the ratio S units / G units in the composition of lignin is meant a decrease or an increase in this ratio. “Wild corn” is understood to mean a corn having digestible characteristics, a ratio between the S units and the G units of the lignin, as well as a content of lignin in the plant similar, or even identical, to those of the line. corn designated "WT" in the examples, which is a line available from the National Institute for Agronomic Research (INRA) under the name I ₂ .

According to the invention, the improved digestibility characteristics of corn, or of these derivative products, are obtained by the modulation of the synthesis of methyltransferase COMT, which modulation of synthesis induces:

(i) the modification of the ratio S units / G units in the composition of lignin; and or

(ii) modification of the lignin content in the corn plant, and the products derived therefrom. According to the invention, the modulation of the synthesis of methyltransferase COMT can be supplemented by the simultaneous modulation of the synthesis of a second enzyme involved in the lignin biosynthesis pathway, preferably a second enzyme involved in the pathway biosynthesis of lignin in corn as described in the prior art and in particular illustrated in the diagram of the

Figure 1.

The decrease in the synthesis of methyltransferase COMT can be obtained by the transformation of maize plants, in particular wild maize plants, by an antisense polynucleotide inhibiting the translation of the corresponding messenger RNA. This reduction can also be obtained by a co-suppression phenomenon, as described for example by Napoli et al. (1990) or Carvalho Niebel et al. (1995).

Conversely, the increase in the synthesis of methyltransferase COMT can be obtained by transformation of corn plants, in particular wild corn plants, with a polynucleotide encoding this enzyme.

Whichever polynucleotide is used, it preferably comprises at least one regulatory sequence allowing its expression in a corn cell. According to the invention, a reduction in the ratio S units / G units is obtained at least by specific inhibition of the COMT activity. It can be supplemented by increasing or reducing the synthesis of other enzymes involved in the biosyrithesis of S units and G units of corn lignin, increase or reduction of activity obtained mainly by overexpression or on the contrary inhibition of expression of genes encoding these other enzymes.

An inhibition of the COMT activity of maize is mainly obtained according to the invention by the use of oligonucleotides which specifically inhibit the transcription or the translation of the gene coding for this enzyme.

The nucleic acid encoding the COMT of maize is referenced as the sequence SEQ ID No. 1 of the sequence listing.

The subject of the invention is the use of a polynucleotide modulating the synthesis of corn COMT methyltransferase encoded by the nucleotide sequence SEQ ID No. 1, in a process for obtaining corn plants with improved digestibility characteristics having a ratio between the units S and G of the modified lignin and / or having a modified lignin content remaining compatible with the normal development of the plant. It also relates to a process for obtaining maize plants having improved digestibility characteristics, having a ratio between the units S and G of the modified lignin and / or whose lignin content is modified and compatible with normal development. of the plant, said method comprising a step of transforming a corn host cell with a polynucleotide chosen from the following polynucleotides: a) an antisense polynucleotide specifically inhibiting the expression of the gene encoding COMT methyltransferase corn; b) a polynucleotide encoding COMT methyltransferase from corn. Preferably, the antisense polynucleotide is the polynucleotide of sequence SEQ ID No. 2.

Preferably, the polynucleotide encoding corn COMT methyltransferase is the polynucleotide of sequence SEQ ID No. 1.

The antisense polynucleotide or the polynucleotide encoding corn methyltransferase COMT are preferably inserted into an expression cassette allowing their functional expression in corn. Such an expression cassette is advantageously introduced into an expression vector suitable for transforming corn cells. According to the method of the invention, it is possible to use a second polynucleotide modulating the activity of an enzyme involved in the pathway for biosynthesis of the S and G units of lignin, other than the methyltransferase COMT.

The invention also relates to a corn plant transformed according to the above process, which has improved digestibility characteristics, as well as to any part of the transformed corn plant, in particular root, seed, leaf, flower and stem.

It also relates to the transformation products obtained from the transformed corn plants defined above, in particular fodder and purified lignin.

DESCRIPTION OF THE FIGURES

Figure 1: Biosynthetic pathway for lignin monomers (Guo et al.,

2001). The "metabolic network" described in the diagram includes the results of recent studies suggesting unexpected substrate specificities of the enzymes F5H and COMT (Humphreys et al. 1999; Osakabe et al. 1999; Li et al. 2000). The framed pathway (guaiacyl [G] unit) represents the main reactions leading to lignins G. The framed pathway (syringyl [S] unit) represents the preferred biosynthetic pathway for lignins S, 4CL, 4-coumarate coenzyme A ligase; CAD, cinnamyl alcohol dehydrogenase; CCR, cinnamyl coenzyme A reductase.

Figure 2: Principle of the method used to assess the impact of the transformation targeting COMT activity on the structure of lignins. The units H (R ₁ = R ₂ = H), G (Rt≈OCHs, R ₂ = H), S (Ri≈Rz≈OCHs) and 5-OH G (R-ι = OCH ₃ , R ₂ = OH ) engaged in -0-4 bonds generate specific thioethylated monomers. After silylation (BSTFA), these monomers are analyzed by CPG-SM (electronic impact). Chromatograms reconstructed on benzyl ions specific to each of them makes it possible to evaluate unequivocally and without interference from the other constituents the relative frequency of the units having generated these monomers.

Figure 3: Chromatographic profiles in CPG-SM of the control lines and under-expressing the COMT: profiles reconstructed on the specific ions of each type of monomer.

Figure 4: HPLC analysis of products from mild alkaline hydrolysis (1 M NaOH, 2.5h, 40 ° C) of extracted corn samples (stems + leaves). P-coumaric acid E and Z: peaks 1 and 2. Ferulic acid E and Z: peaks 3 and 4. Standard (ethylvanillin): peak 5. These profiles illustrate that the contents of PC (p-coumaric acid) decrease in both ASCOMT1.2 and ASCOMT3.2 transgenic lines compared to the WT control.

Figure 5: Schematic representation of the constructions of the plasmids described in Example 1.

Figure 6: Figure 6A shows a Northern blot analysis of the 20-day C-OMT 225 corn line.

Figure 6B shows a comparative Southern blot analysis between the antisense line 225 and the line, using four separate restriction enzymes, respectively: AcoRV, Kpnl, EcoRI and SacI.

Figure 7: Screening of a population of transgenic maize plants, transformed respectively with the plasmids pProsper + pBar, pProsper-Bar, pJim + pBar2 and pALIX + pBar2.

The ordinate axis represents the COMT activity found in the corn plants analyzed, expressed in cpm / mg of protein.

The histogram bars each represent a tested transgenic plant.

Figure 8: cribro-vascular bundles of adult control corn leaves (I ₂ ) and C-OMT antisense (225) stained according to Maϋle's method. We see that the coloration is less intense in the sclerenchyma and in the xylem of 225; this observation suggests a decrease in S units in the lignins of line 225.

DETAILED DESCRIPTION OF THE INVENTION

It has been shown according to the invention that, surprisingly, the induction of the modulation of the synthesis of an enzyme involved in the biosynthesis pathway of corn lignin, methyltransferase COMT, also designated COMT, causes a modification of the S units / G units ratio in the composition of lignin and / or of the lignin content, compared to that found in wild corn, and that the modification of the S units / G units ratio and / or of the content Lignin gave the corn plant, as well as its transformation products, improved digestibility characteristics. By “improved digestibility characteristics” of a corn plant according to the invention, it is meant that, compared with a variety of wild corn plant, for example the aforementioned variety of corn I ₂ , the corn plant obtained according to the invention has a higher digestibility index value "dndf" than the digestibility index value calculated from a corn plant of the wild variety.

According to the invention, the value of the digestibility index "dndf" is calculated from "ndf (neutral detergent fiber)" whose method of obtaining is described by Van Soest (1967) and "dms (enzymatic solubility Aufrère) ”, the method of obtaining which is described by Aufrère (1983), according to the formula dmdf = 100 * [dms - (100 - ndf)] / ndf given by Struik (1985). The material used for the establishment of the ndf is a lyophilized powder of whole plants harvested at the flowering stage.

Preferably, a corn plant obtained according to the invention has improved digestibility characteristics if the value of the “dndf” index is at least 80.

Preferably, a corn plant according to the invention has a Klason lignin content, measured from the stems and leaves, which is less than 8.5, preferably less than 8, 7.5, to, 7.0 , at 6.9 or 6.8, and preferably less than 6.7. A preferred corn plant according to the invention has a Klason lignin content of between 6.1 and 6.6.

To measure the Klason lignin content, a person skilled in the art can advantageously refer to the technique described in Example 9. It is shown for the first time according to the invention, surprisingly: a) that the inhibition of COMT methyltransferase corn enzyme allowed (i) to reduce the ratio S units / G units of lignin in this plant and (ii) to reduce the lignin content in this plant; and b) that the reduction of the S units / G units ratio of lignin and / or of the lignin content in maize made it possible to obtain maize plants having improved digestibility characteristics, compared to wild maize.

Taking advantage of these unexpected results, the applicant has developed a process for transforming corn plants in which the transformed corn plants have a ratio S units / G units of lignin and / or a modified lignin content, relative to that found in wild corn plants.

Preferably, the S units / G units ratio is lower than the S units / G units ratio found in a wild corn plant.

More specifically, it is shown according to the invention that transformed corn plants having improved digestibility characteristics can be obtained using an antisense polynucleotide. The subject of the invention is the use of a polynucleotide modulating the synthesis of corn COMT methyltransferase encoded by the nucleotide sequence SEQ ID No. 1, in a process for obtaining corn plants with improved digestibility characteristics having a ratio between the units S and G of the modified lignin and / or having a modified lignin content remaining compatible with the normal development of the plant.

Inhibition of the synthesis of methyltransferase COMT

According to a first embodiment, said polynucleotide inhibits the synthesis of COMT methyltransferase from corn in the plant, when it is introduced by transformation into the cells of said plant. Said antisense polynucleotide consists of a polynucleotide coding for an antisense RNA hybridizing with the messenger RNA constituting the transcript of the gene coding for methyltransferase COMT in maize.

Inhibition of the synthesis of methyltransferase COMT can be obtained:

(i) either by transforming the corn cells with an antisense polynucleotide, as defined above, placed under the control of a functional regulatory sequence in corn, said regulatory sequence possibly comprising a strong constitutive promoter and, if necessary, also transcription activator sequences

(functional enhancer sequences in corn), allowing a high level of expression of the copy number of the mRNA of the antisense methyltransferase COMT;

- (ii) either by stably introducing into the corn cells a plurality of copies of the antisense polynucleotide as defined above;

- (iii) either by a combination of (i) and (ii), as described in the examples.

A person skilled in the art can synthesize any antisense polynucleotide inhibiting the synthesis of methyltransferase COMT using his general technical knowledge, from a nucleotide sequence coding for COMT, for example the sequence SEQ ID No. 1.

For the synthesis of a sense polynucleotide or an antisense polynucleotide which specifically inhibits the transcription or translation of a gene in a plant, those skilled in the art will advantageously refer to the review article by Matzke et al. (2001) as well as the content of the articles referenced in this review article.

Even more specifically, for the construction or synthesis of a sense polynucleotide or of an antisense polynucleotide which specifically inhibits the transcription or translation of a gene coding for an enzyme involved in the biosynthesis of lignins in plants, in particular the COMT of certain plant species, those skilled in the art can advantageously refer to the articles by Baucher et al. (1999), de Zhong et al. (2000), de Pinçon et al. (2001a), de Blee et al. (2001), de Pinçon et al. (2001 b) or Lapierre et al. (1999). The antisense polynucleotide can also consist of the cDNA obtained from the mRNA constituting the transcription product of the gene coding for methyltransferase COMT.

It can also consist of a nucleotide fragment of this cDNA, preferably a nucleotide fragment of at least 100 consecutive bases of this cDNA, this length of the nucleotide fragment being sufficient to allow its specific hybridization with the methyltransferase COMT mRNA of maize and thus prevent its translation into protein. According to a first preferred aspect, the antisense polynucleotide comprises the nucleotide sequence SEQ ID No. 2.

According to a second preferred aspect, the antisense polynucleotide consists of the antisense polynucleotide of sequence SEQ ID No. 2.

Increase (overproduction) of methyltransferase COMT

According to a second embodiment, said polynucleotide increases the synthesis of methyltransferase COMT in the plant, when it is introduced by transformation into the cells of said plant. According to this second embodiment, a polynucleotide encoding COMT methyltransferase from corn is used, which causes a higher production of this enzyme, when it is introduced by transformation into the plant.

The synthesis or isolation of a polynucleotide encoding COMT methyltransferase from corn can be carried out by a person skilled in the art, using his general knowledge in molecular biology, in particular thanks to his knowledge of the nucleotide sequence SEQ

ID # 1.

The higher production of methyltransferase COMT can be obtained: - (i) either by transforming corn cells with a polynucleotide encoding methyltransferase COMT placed under the control of a functional regulatory sequence in corn, said regulatory sequence being able to include a strong constitutive promoter and, where appropriate, also transcription activator sequences (functional “enhancer” sequences in corn), allowing a high level of expression of the methyltransferase COMT;

- (ii) either by stably introducing into the corn cells a plurality of copies of the polynucleotide coding for methyltransferase COMT;

- (iii) either by a combination of (i) and (ii).

According to a first preferred aspect, the polynucleotide coding for methyltransferase COMT comprises the nucleotide sequence SEQ ID No. 1. According to a second preferred aspect, the polynucleotide coding for methyltransferase COMT consists of the nucleotide sequence SEQ ID No. 1.

PROCESSES FOR OBTAINING CORN PLANTS WITH IMPROVED DIGESTIBILITY CHARACTERISTICS.

A subject of the invention is also a process for obtaining maize plants having improved digestibility characteristics, having a ratio between the units S and G of the modified lignin and / or the lignin content of which is modified and compatible with the normal development of the plant, said method comprising a step of transforming a corn host cell with a polynucleotide chosen from the following polynucleotides: a) an antisense polynucleotide specifically inhibiting the expression of the gene encoding COMT methyltransferase corn; b) a polynucleotide encoding COMT methyltransferase from corn.

According to a first embodiment of the above method can be characterized in that it comprises the following steps: a) transforming a corn host cell with an antisense polynucleotide specifically inhibiting the transcription or translation of the gene coding for the methyltransferase COMT of But.; b) regenerating an entire plant from the recombinant host cell obtained in step a);

According to a first aspect, the modification of the lignin composition of the transformed corn plants capable of being obtained by the above process consists in reducing the content of S units relative to the content of G units in corn lignins, so as to obtain a lower S / G ratio than that of wild corn plants.

Preferably, the S / G ratio is less than 40/60.

According to a second embodiment of the above method, said method is characterized in that it comprises the following steps: a) transforming a corn host cell with a polynucleotide encoding corn COMT methyltransferase COMT; b) regenerating an entire plant from the recombinant host cell obtained in step a); Preferably, the polynucleotide encoding methyltransferase

COMT of maize comprises the nucleotide sequence SEQ ID N ° 1.

According to a particular embodiment, the process for obtaining transformed corn plants according to the invention is characterized in that it comprises the following additional step: c) selecting the corn plants having integrated into their genome at least one copy of the antisense polynucleotide or at least one copy of the polynucleotide encoding COMT methyltransferase from maize.

Expression cassettes comprising an antisense polynucleotide or a polynucleotide encoding COMT methyltransferase from maize

Advantageously, the antisense polynucleotide or the polynucleotide encoding COMT methyltransferase from corn is integrated into an expression cassette also comprising a nucleotide sequence regulating the expression of said polynucleotide in corn cells. Advantageously, the antisense polynucleotide or the polynucleotide coding for methyltransferase COMT is integrated into an expression cassette also comprising a nucleotide sequence with a transcription terminator function.

The expression cassette defined generally above may also be designated “DNA construct” or “DNA construct” in the present description.

According to yet another aspect, the antisense polynucleotide or the polynucleotide coding for methyltransferase COMT, where also the expression cassette in which is inserted respectively one or the other of these polynucleotides, is artificially inserted into a host cell of Agrobacterium tumefaciens.

The DNA constructs also include a gene promoter sequence and a terminator sequence operably linked to the DNA sequence to be transcribed. The gene promoter is generally located at the 5 'end to allow initiation of transcription of the DNA sequence. The promoter sequences are located in the 5 'non-coding regions of the genes but also sometimes in the introns (Luehrsen K ,. 1991) or in the coding regions such as, for example, the promoter of the PAL gene in tomatoes (Bloksberg, 1991). When the construct includes an open sense reading phase, it is necessary to use a full length cDNA so that the protein can be translated. When the construct comprises an open reading phase in antisense or a non-coding region, it is not essential to use a full length cDNA, a partial sequence may suffice.

Promoters

Among the transcription promoters which can be used, mention may be made in particular of the so-called constitutive promoters, the so-called inductive promoters as well as the specific tissue promoters.

A constitutive promoter allows a strong expression of the transcript in all the tissues of the regenerated plant and will be active in most environmental conditions, and in all the stages of cellular transformation and differentiation. For example, the main dual component promoters are:

- the 35S promoter, or advantageously the double constitutive promoter 35S (pd35S) of CaMV, described in the article by Kay et al., (1987) - the rice actin promoter followed by the rice actin intron contained in the plasmid pAct1-F4 described by Me Elroy et al. (1991)

- the corn ubiquitin 1 promoter (Christensen et al., 1996) and other regions initiating the transcription of genes of variable plants known to those skilled in the art. Alternatively, it may be advantageous to use an inducible transcription promoter sequence capable of being controlled by environmental conditions or the development stage, such as, for example, the phenylalanine ammonia lyase (PAL), HMG-CoA reductase (HMG) promoters. , chitinases, glucanases, proteinase inhibitors (PI), genes of the PR1 family, nopaline synthase (nos) or the vspB gene (US-5,670,349), all of these promoters being recalled with references corresponding publications by Table 3 of US Patent ~ 5,670,349. Another category of promoters which can advantageously be used to carry out the process which is the subject of the invention groups together specific tissue promoters, in particular they may be promoters of genes for the lignin biosynthesis pathway or for example the promoter of the alcohol dehydrogenase which is expressed specifically in vascular tissue.

Mention may also be made of specific seed promoters (Datla, R. et al., 1997), in particular the promoters of napine (EP-0.255.378), glutenin, heliantinin (WO-92/17580) , albumin (WO-98/45460), oleosin (WO-98/45461), IΑTS1 or IΑTS3 (WO-99/20775).

Terminating sequences

The terminator sequence used in the constructions which are the subject of the invention is located at the 3 ′ end of the sequence to be transcribed. It can come from the same gene as the promoter sequence or from a different gene.

Among the transcription terhninators which can be used, there may be mentioned:

the polyA 35S terminator of the cauliflower mosaic virus (CaMV) described in the article by Franck et al., (1980), or

the polyA NOS terminator, which corresponds to the 3 ′ non-coding region of the nopaline synthase gene of the Ti plasmid of Agrobacterium tumefaciens nopaline strain. Selection marker

The constructs also include a selection marker for selecting the plant cells transformed by these constructs. These selection markers are well known to those skilled in the art, in particular they confer resistance to one or more toxins, for example resistance to the herbicide BASTA®. Furthermore, the transformation of plant cells by the constructions which are the subject of the invention can be controlled by other techniques well known in the art such as Southern and Western Blots.

Making an expression cassette

Techniques for operably linking all components of these constructs are well known to those skilled in the art and include the use of synthetic cloning sites comprising one or more endonuclease restriction sites as described for example by Maniatis et al . (1989). The DNA constructs of the present invention can be linked to a vector having at least one replication system, for example with E. Coli, after each manipulation, it is possible to clone and sequence the construct obtained in order to verify the correct handling.

The invention relates to the constructions as described above, and comprising at least one sequence chosen from the sequences SEQ ID No. 1 or 2 linked operatively upstream to a promoter and downstream to a terminator. Most preferably, the expression cassette comprises the promoter of the alcohol dehydrogenase specific for the vascular system, the cDNA of methyltransferase (COMT or AldOMT) and the terminator of Nopaline synthase, as shown in FIG. 'example 1.

Expression vectors

The expression cassettes or the DNA constructs as defined above can be included in expression vectors which can be used to transform an Agrobacterium tumefaciens cell or a corn host cell. The preferred bacterial vectors according to the invention are for example the vectors pBR322 (ATCC No. 37 017) or also the vectors such as pAA223-3 (Pharmacia, Uppsala, Sweden) and pGEM1 (Promega Biotech, Madison, Wl, United States ). Mention may also be made of other commercially available vectors such as the vectors pQE70, pQE60, pQE9 (Quiagen), psiX174, pBluescript SA, pNH8A, pMH16A, pMH18A, pMH46A, pWLNEO, pSV2CAT, pOG44, pXTI and pSG (Stratagene).

They may also be vectors of the Baculovirus type such as the vector pVL1392 / 1393 (Pharmingen) used to transfect the cells of the line Sf9 (ATCC No. CRL 1711) derived from Spodoptera frugiperda.

Preferably, use will be made of vectors specially adapted for the expression of sequence of interest in plant cells, such as the following vectors: - vector pBIN19 (BEVAN et al.), Marketed by the Company

CLONTECH (Palo Alto, California, USA);

- vector pB1 101 (JEFFERSON, 1987), sold by the company CLONTECH;

- vector pBI121 (JEFFERSON, 1987), sold by the company CLONTECH;

- vector pEGFP; Yang et al. (1996), marketed by the company CLONTECH;

- vector pCAMBIA 1302 (HAJDUKIIEWICZ et al., 1994)

- intermediate and superbinary vectors derived from the pSB12 and pSB1 vectors described by Japan Tobacco (EP-0.672.752 and Ishida et ai,

1996).

A first very preferred vector is the plasmid pAlix represented in FIG. 5, into which has been inserted the antisense polynucleotide of sequence SEQ ID No. 2 inhibiting the synthesis of methyltransferase COMT from maize.

A second very preferred vector is the plasmid pProsper represented in FIG. 5, into which has been inserted the polynucleotide of sequence SEQ ID No. 1 coding the methyltransferase COMT of corn. Processing of corn plants

An increase in the synthesis of the S and / or G units of lignin and / or of the lignin content in the plant can be obtained by integrating in the open reading phase a DNA construct in sense orientation, in particular the sequence SEQ ID # 1. The transformation of a target plant with such a construction results in an increase in the number of copies of the transcript and therefore in an increase in the quantity of enzyme. For example, an increase in the synthesis of S units can be obtained by integrating into the genome of the target plant the sense sequence of O methyltransferase encoded by SEQ ID No. 1.

A reduction in the synthesis of S and / or G units of lignin and / or of the lignin content in the plant can be obtained by introducing into the open reading phase a DNA construct in antisense orientation, in particular any or part of the sequence SEQ ID n ° 2. The transcribed RNA is complementary to the endogenous mRNA sequence, which will have the consequence of reducing the number of copies of the transcript and therefore of reducing the quantity of enzyme. For example, a decrease in the synthesis of S units can be obtained by integrating into the genome of the target plant the antisense sequence of methyltransferase COMT coded by SEQ ID No. 2. The DNA constructs can also comprise a nucleotide sequence comprising a non-coding region of the methyltransferase COMT gene. Examples of non-coding regions which can be used in such constructs include introns and 5 'or 3' non-coding sequences. Transformation of target plants with such DNA constructs can lead to a reduction in the synthesis of S and / or G units of lignin and / or the lignin content by the co-suppression process, as has described for example Napoli et al. (1990) or Carvalho Niebel et al. (1995).

The regulation of the synthesis of S and / or G units of lignin and / or of the lignin content can also be carried out by inserting said sequences in whole or in part (for example DNA or RNA) in ribozyme constructs (Haseloff and al., 1988). Use of a second polynucleotide modulating the synthesis of an enzyme involved in the biosynthesis of lignin, other than methyltransferase COMT

According to yet another aspect of the process for obtaining maize plants having improved digestibility characteristics, as defined above, the effect of the antisense polynucleotide or of the polynucleotide encoding the methyltransferase COMT of maize can be completed by the insertion in the genome of the same corn plant of a second polynucleotide derived from the gene encoding an enzyme involved in the lignin biosynthesis pathway, other than the methyltransferase COMT.

Thus, the above method can also be characterized in that in step a), the corn host cell is also transformed with at least one second polynucleotide, specifically modulating the expression of an enzyme involved in the pathway biosynthesis of lignins, other than corn COMT methyltransferase.

According to this particular aspect, said method is characterized in that the second polynucleotide is chosen from: a) an antisense polynucleotide specifically inhibiting the expression of the gene coding for the enzyme involved in the lignin biosynthetic pathway, other than the methyltransferase COMT of But ; b) a polynucleotide encoding the enzyme involved in the lignin biosynthesis pathway, other than corn COMT methyltransferase.

The enzyme involved in the lignin biosynthesis pathway, other than COMT methyltransferase from corn, is chosen from: (a) the CAD enzyme from corn, the sequence of which is referenced in the GenBank database under the access number Y13733, (b) the corn CCR1 enzyme whose sequence is referenced in the GenBank database under access number X98083 and (c) the F5H enzyme from Arabidopsis thaliana whose sequence is referenced in the base GenBank data under access number U38416.

The enzyme involved in the lignin biosynthesis pathway, other than COMT methyltransferase from corn, can also be chosen from (d) the CCoAOMTI enzyme from corn, the sequence of which is referenced in the GenBank database under the number d access AJ242980 and (e) the corn CCoAOMT2 enzyme whose sequence is referenced in the GenBank database under the access number AJ242981.

Once obtained, the constructs serve to transform plant cells. Advantageously, the transformation of plant cells can be carried out by transferring the above-mentioned vectors into plant protoplasts, in particular after incubation of the latter in a polyethylene glycol solution in the presence of divalent cations (Ca 2+) (Schocher ef al., 1986).

The transformation of plant cells can advantageously be carried out by electroporation, in particular according to the method described in the article by Fromm et al. (1985).

The transformation of plant cells can also be carried out by using a particle gun allowing the projection, at very high speed, of metallic particles covered with the DNA sequences of interest, thus delivering the sequences inside the cell nucleus. (Fromm et al. 1990, Finer et al., 1992).

Another method of transforming plant cells is that of cytoplasmic or nuclear micro-injection (Neuhaus et al., 1987). One of the methods of transformation of plant cells which can be used in the context of the invention is the infection of plant cells by a bacterial cell host comprising the vector containing the sequence of interest. Advantageously, the abovementioned cell host used is Agrobacte um tumefaciens, in particular according to the method described in the article by An et al. (1986), or even Agrobacterium rhizogenes, in particular according to the method described in the article by Guerche et al. (1987).

Preferably, the transformation of plant cells is carried out by the transfer of the T region of the extrachromosomal circular plasmid inducing Ti tumors of Agrobactenum tumefaciens, using a binary system (Watson et al., 1994). To do this, two vectors are constructed. In one of these vectors, the T-DNA region was deleted, with the exception of the right and left edges, a marker gene being inserted between them to allow selection in plant cells. The other partner of the system binary is a helper Ti plasmid, a modified plasmid which no longer has T-DNA but still contains the vir virulence genes necessary for the transformation of the plant cell. This plasmid is maintained in Agrobacterium. According to a preferred mode, the method described by Ishida et al. (1996) can be applied for the transformation of monocots, in particular maize.

According to another protocol, the transformation is carried out according to the method described by Finer et al. (1992) using the tungsten or gold particle gun.

According to yet another aspect, the process for obtaining transformed corn plants according to the invention is characterized in that it comprises the following additional steps: d) crossing between them of two transformed plants as obtained in step b) or in step c) with a second corn plant; e) selection of the plants homozygous for the transgene or the transgenes.

According to yet another aspect, the process for obtaining transformed corn plants according to the invention is characterized in that it comprises the following additional steps: f) crossing of a transformed corn plant obtained in step c) with a second corn plant; g) selection of the corn plants resulting from the crossing of step f) and having conserved the transgene. One of the embodiments of the process which is the subject of the invention relates to obtaining transgenic maize plants from plant cells transformed with a construct comprising at least one sequence chosen from the sequences SEQ ID No. 1 or SEQ ID No. 2 .

Host cells transformed by an antisense polynucleotide or by a polynucleotide coding for methyltransferase COMT and transformed corn plants obtained from the transformed host cells.

The subject of the invention is also a corn host cell or an Agrobacterium tumefaciens cell transformed with: a) either an antisense polynucleotide inhibiting the synthesis of methyltransferase COMT in a corn cell; b) or a polynucleotide coding for methyltransferase COMT, said polynucleotide (a) or said polynucleotide (b) being preferably placed under the control of a functional regulatory sequence in a corn cell, for example in an expression cassette or else in an expression vector as defined above.

The present invention relates to the cells of transformed plants thus obtained. In particular, the corn cells having stably integrated into their genome the constructs comprising all or part of the sequences SEQ ID No. 1 or No. 2.

The cells transformed by the constructions which are the subject of the present invention are selected by means of the selection marker, for example resistance to BASTA®. The transgenic cells are then cultivated on an appropriate medium to regenerate whole plants using methods well known to those skilled in the art. In the case of protoplasts, the cell wall can reform under the effect of appropriate osmotic conditions. In the case of seeds or embryos, a medium favorable to the germination or the initiation of calluses is used. For the explants, the medium used must allow regeneration.

The principle of in vitro regeneration of corn plant cells is well known to those skilled in the art. The plants are regenerated from calluses obtained after culturing anther cells, pollen grains, embryos or protoplasts. The cells are cultivated on agar nutritive media adapted to each cell type under sterile conditions in petri dishes or culture tubes in a climatic chamber under favorable environmental conditions (suitable temperature and brightness). After transplanting, the vitroplants are transplanted to the greenhouse.

In particular, according to one aspect of the invention, the corn plants offering better digestibility and whose content of S and / or G units is modified, in particular having a S / G ratio lower than that of plants whose ratio n is not modified can be obtained after hybridization with an individual whose activity of methyltransferase COMT is modified by the principles of varietal selection well known to those skilled in the art.

A person skilled in the art can also employ the methods of vegetative multiplication from corn plants whose methyltransferase COMT activity is modified, in particular in order to modify the ratio in S units / G units and / or the lignin content. These methods can advantageously include certain steps in vitro. These can be stages of regeneration of cells having a modified COMT methyltransferase activity. It can be methods of tissue culture, micro-cuttings, culture of meristems, culture of embryos, corn plants having a modified methyltransferase COMT activity. These can be methods of regenerating protoplasts from corn plants having a modified COMT methyltransferase activity or from fusion with protoplasts from corn plants having a modified COMT methyltransferase activity.

The subject of the invention is also a transformed corn plant, capable of being obtained by the process as defined above, which is characterized by digestibility characteristics. improved.

According to a first characteristic which can define it, a corn plant transformed in accordance with the invention has improved digestibility characteristics and a value of the index “dndf” of at least 80. According to a second characteristic which can define it, a plant of corn according to the invention has a Klason lignin content, measured from the stems and leaves, which is less than 8.5, preferably less than 8, 7.5, to, 7.0, to 6, 9 or 6.8, and preferably less than 6.7. A preferred corn plant according to the invention has a Klason lignin content of between 6.1 and 6.6.

According to a third characteristic which can define it, a corn plant transformed according to the invention has an S / G ratio of lignin of less than 40/60. The invention also relates to a part of a transformed corn plant as defined above, for example a root, a seed, a leaf, a stem or a flower.

It also relates to isolated corn cells obtained from a transformed corn plant as defined above.

The invention also relates to a recombinant expression vector comprising a polynucleotide chosen from: a) an antisense polynucleotide specifically inhibiting the expression of the gene encoding COMT methyltransferase from corn; b) a polynucleotide coding for methyltransferase COMT; said polynucleotide being placed under the control of a functional regulatory sequence in corn.

The present invention also relates to a DNA construct comprising a sense polynucleotide or an antisense polynucleotide specifically inhibiting the transcription or translation of the COMT gene of sequence SEQ ID No. 1, placed under the control of a functional promoter in maize .

It also relates to a DNA construct comprising a polynucleotide coding for methyltransferase COMT, placed under the control of a functional promoter in corn to increase the expression of said enzyme.

The invention also relates to a transformation product obtained from a transformed corn plant or part of a transformed corn plant, as defined above. Such a transformation product is characterized in particular in that the lignin which it contains has a modified ratio between the S units and the G units. Preferably, the S / G ratio is less than 40/60. Said transformation product includes animal feed, obtained from corn plants whose contents in S units or in G units are modified and / or whose lignin content is modified.

The subject of the invention is also a lignin purified from a transformed corn plant or a part of a corn plant as defined above, characterized in that it has in particular a relationship between the units S and the G units which is modified, compared to the S / G ratio found in lignin from wild corn plants. Preferably, the lignin purified above is characterized in that the S / G ratio is less than 40/60.

The present invention is illustrated, without however being limited, to the following examples.

EXAMPLES

Example 1: Construction of transformation vectors:

Several vectors have been constructed in order to obtain the transformation vector.

- pProsper Plasmid [Figure 5]

A 0.26 Kpb Pstl-Hindlll fragment corresponding to the terminator of the nopaline synthase gene (T-Nos) is subcloned in pUC18 (Yannisch-Perron C. et al., 1985) between the PstI and Hindlll sites, thus giving the vector pUC18-Nos. Plasmid pMC1 (Collazo P. et ai, 1992), corresponding to the c-OMT cDNA (1.4 kb) inserted at the pst1 site of pUC18. PCR amplification is carried out on this plasmid with the oligonucleotides TOM22 (5'-CTGCTGGAGGTGCTGCAGAAG-3 '- SEQ ID N ° 3) and TOM23 (5'-CTCCTTGCCCCCGGGGTTGTG-3 - SEQ ID N ° 4'), allowing respectively to introduce the PstI and Smal sites, 5 ′ and 3 ′ of a 0.85 Kpb fragment between the positions 0.17 and 1.04 Kpb of the cDNA. This fragment is then subcloned into the vector pGemT (Promega), and the plasmid obtained is digested with PstI and SmaI. The released fragment is cloned into pUC18-Nos at the PstI and SmaI sites, thus generating the plasmid pTMO-Nos.

The plasmid pBARGUS (Fromm et a /., 1990) contains a CaMV35S-intron Adhl-BAR-Tnos promoter cassette and an AdhI-Intron AdhI-GUS-Tnos promoter cassette. The latter is released by EcoRI and Hindlll digestion. In a second step, partial EcoRI / Pvull digestion of the same fragment makes it possible to release a 1.75 Kpb fragment corresponding to the Adhl promoter coupled to the Adhl intron. This fragment is then subcloned into pTMO-Nos between EcoRI and Smal. The plasmid obtained is pProsper. - pAlix plasmid [Figure 5]

The PMC1 plasmid is digested with HindIII and Xbal to release the full length (1.4 Kbp) cDNA from C-OMT. The plasmid pActine-BAR (Wu, (Cornell University, New York) consists of the plasmid psP72 containing an actin - Intron actin - BAR - Tnos promoter cassette. This plasmid is digested with Hindlll and Xbal to remove the BAR fragment, then the fragment 1.4 Kbp Hindlll-Xbal of C-OMT is subcloned between these two sites, thus giving the plasmid pAlix.

- pBar2 plasmid [Figure 5]

The plasmid pBARGUS is digested with HindIII, to release the promoter cassette CaMV35S - Intron Adhl - BAR - Tnos of 2.13 Kpb. The cassette thus released is subcloned into pUC18 at the Hindlll site, to give the plasmid pBar2.

- pBar Plasmid [Figure 5]

The construction of the plasmid pBar is described by Peter Eckes (Biology Research Center H 872 N, Hoechst AG 6230 Frankfurt 80 / EP-0.275.957).

Example 2 Transformation by Agrobacterium tumefaciens

The transformation technique described by Ishida et al. (1996) is used from immature embryos 10 days after fertilization. All the media used are referenced in the cited reference.

The transformation begins with a co-culture phase where the immature embryos of the corn plants are brought into contact for at least 5 minutes with Agrobacterium tumefaciens LBA 4404 containing the superbinary vectors. The superbinary plasmid is the result of homologous recombination between an intermediate vector carrying the T-DNA containing the gene of interest and / or the selection marker derived from the plasmids described in the previous examples, and the vector pSB1 from Japan. Tobacco (EP 672 752) which contains: the virB and virG genes of the plasmid pTiBo542 present in the supervirulent strain A281 of Agrobacterium tumefaciens (ATCC 37349) and a homologous region found in the intermediate vector allowing this homologous recombination.

The embryos are then placed on LSAs medium for 3 days in the dark and at 25 ° C. A first selection is made on the transformed calluses: the embryogenic calluses are transferred to LSD5 medium containing phosphinotricin at 5 mg / l and cefotaxime at 250 mg / l (elimination or limitation of contamination by Agrobacterium tumefaciens). This stage is carried out for 2 weeks in the dark and at 25 ° C. The second selection step is carried out by transfer of the embryos which have developed on LSD5 medium, on LSD10 medium (phosphinotricin at 10 mg / l) in the presence of cefotaxime, for 3 weeks under the same conditions as above. The third selection step consists in excising the type I calluses (fragments of 1 to 2 mm) and transferring them 3 weeks in the dark and at 25 ° C to LSD 10 medium in the presence of cefotaxime.

The regeneration of the seedlings is carried out by excising the type I calluses which have proliferated and by transferring them to LSZ medium in the presence of phosphinotricin at 5 mg / l and cefotaxime for 2 weeks at 22 ° C. and under continuous light.

The regenerated seedlings are transferred to RM + G2 medium containing 100 mg / l of Augmentin for 2 weeks at 22 ° C and under continuous illumination for the development stage. The plants obtained are then transferred to the phytotron for their acclimatization.

Example 3: Transformation by bombardment

Calluses of genotype I ₂ , 2 months old, obtained after the cultivation of immature embryos on a callogenesis medium are used for transformation. A plasmolyzing treatment makes it possible to reduce the volume of the vacuole and thus limits the cell bursts during firing (pretreatment of 4 hours and a post-treatment of 16 hours on a Sorbitol 0.2M and Mannitol 0.2M medium).

The plant material is distributed evenly across all of the boxes.

15 mg of sterile tungsten microbeads are mixed in 150 μl of sterile milliQ water.

For 5 shots, the following mixture is prepared:

25μl of tungsten microbeads,

2.5 μl of DNA of the plasmid carrying the gene of interest (at 1 μg / μl),

2.5 μl of DNA of the plasmid carrying the selection gene (at 1 μg / μl), - 25 μl of CaCl2 at 2.5M,

10 μl of 0.1 M spermidine. This mixture is left to settle in ice for at least 5 minutes.

Five Petri dishes containing the material to be bombarded are placed on Agar dishes at 15 g / L.

50 μl of supernatant from the decanted tube is eliminated and the remaining mixture is well vortexed, then 2 μl of it is placed on the grid of the sterile syringe, which is screwed onto the support in the barrel.

A sterile gauze filter is placed on the petri dish which is placed 19 cm in the barrel enclosure. The vacuum is pushed inside the enclosure at a pressure of 25 mbar

The shooting is started. The helium pressure in the barrel is 8 bars.

The bombarded plant material is returned to its original petri dish (Mannitol / Sorbitol). The boxes are placed at 26 ° C and in the dark for 16 hours for a post-bombardment plasmolyzing treatment (mannitol / sorbitol).

The selection pressure is applied 16 hours after firing and is maintained at the same concentration of the selective agent (bialaphos at 5 mg / L) during all the regeneration steps. The seedlings are then transplanted into potting soil (small pots) for 1 week then transferred to 20-liter pots (peat-pozzolan mixture) in the transgenic greenhouse or in an air-conditioned room (temperature 24 ° C day / 20 ° C night; photoperiod 16h day / 8h night, humidity 80%). The plants are self-fertilized or crossed.

Example 4 Analysis of the RNAs by Northern Blot and of the DNA DNA by Southern Blot

A. Materials and Methods

The RNAs are isolated by the Extract-all solution (Eurobio), according to the supplier's protocol. Additional precipitation with 0.2 M NaCl with 2 volumes of absolute ethanol is carried out. 10 μg of RNA are separated on formaldehyde gels (Sambrook et a /., 1989). Total genomic DNA is extracted from 1 g of leaves by the method described in Dellaporta et al. (1983). After a phenol-chloroform purification step, 10 μg of DNA are digested with 40 units of restriction enzyme (Promega), and separated on TAE agarose gel at 1% (m / v). The RNAs or DNAs are transferred onto nylon membranes (Hybond-N +, Amersham), and hybridized with DNA probes labeled with ³² P-dCTP by the "prime-a-gene labeling system" kit from Promega. The hybridization signal is determined by contacting the membranes with Kodak Biomax-MS films in an autoradiography cassette.

B. Results

Figure 6A shows a Northern blot analysis of the 20-day C-OMT 225 corn line. RNAs from roots (r), collar (c), and coiled leaves (H) of line 225 and control line I2 were deposited on agarose electrophoresis gel.

After migration and incubation for 10 minutes in a BET solution at 0.1 μg / ml, the gel was photographed under UV in order to estimate the relative amounts of ribosomal RNAs (rRNA). Hybridization of the membrane after transfer with a ³² P-labeled cDNA probe makes it possible to observe a large decrease in C-OMT transcripts in all in all the organs tested (R, C, H) in line 225, compared with I ₂ .

Figure 6B shows a comparative Southern blot analysis between the antisense line 225 and the line I2, using four distinct restriction enzymes, respectively: EcoRV, Kpnl, EcoRI and Sac1.

The probe used is a 1.2 Kb fragment located in the 3 ′ region of the corn C-OMT cDNA.

The bands revealed on the line I ₂ are due to the endogenous gene C-OMT.

The supernumerary bands present for the line 225 correspond to the transgenic sequences: the enzymes EcoRV, Kpnl and Sacl do not cut in the transgene. An EcoRI site is present in the 5 'part of the transgenic construct. Depending on the case, 3 supernumerary bands (EcoRI, EcoRV) or 2 bands (Kpnl, Sacl) appear for the line 225.

The results show that the line 225 has been transformed with 2 or 3 copies of the transgene.

EXAMPLE 5 Enzymatic Analysis of COMT

A. Materials and Methods The protocol used is derived from Atanassova et al. (1995).

Briefly, young plants of 20 days (post-sowing) are harvested by cutting at the level of the collar, then the leaves are eliminated from the aerial part (cut at the level of the ligule). The remaining fragment, about 15 cm long, is fixed in liquid nitrogen and ground in a mortar with a pestle. The powder obtained is homogenized in the buffer extraction Na ₂ HP0 ₄ / NaH ₂ P0 ₄ 0.1 M pH7.5, PVPP 5% (m / v), DTT (10mM) at the rate of 1 mL of buffer for 300 mg of powder. After V_ hour of incubation at 4 ° C, two successive centrifugations (3000 g / 10 min / 4 ° C) are carried out to remove the insoluble material. 40 μL of crude extract thus obtained is added to 960 μL of reaction mixture (Caffeic acid ³ mM, ³ H-SAM 0.1 μCi, SAM 50 μM, DTT 10 mM, Na ₂ HP0 ₄ buffer / NaH ₂ P0 ₄ 0.1 M pH 7.5 qs 960μL) and the whole is incubated for 1 hour at 37 ° C. The reaction is stopped with 100 μL of 9N sulfuric acid and 5 ml of organic scintillant (OCS, Amersham) are added to the mixture. The whole is homogenized to pass the tritiated ferulic acid, product of the reaction, through the organic scintillant. The radioactivity of ferulic acid is quantified with a Beckman scintillation counter. The proteins are assayed according to the method of Bradford (1976) with the reagent of Biorad.

B. Results

Screening of transgenic plants on the basis of C-OMT activity

(figure 7)

1 - Choice of the part of the plant used for the measurement of C-OMT activity

The plants were harvested at a young stage (20 days old after sowing), which implies a short time between sowing and analysis, and which makes it possible to cultivate a large number of plants simultaneously. The stem and the coiled young leaves were used as a mixture for this work. The ligulate leaves have been eliminated.

2 - C-OMT activity in control plants used for screening The line used as a control in screening is I ₂ . The histogram bar of the controls I ₂ results from the average of the results obtained on 12 plants analyzed twice. We have for these controls activity values which vary from 4385 to 7225 cpm / mg of protein, the average being at 5998 cpm / mg with a standard deviation of 1039. This first result makes it possible to estimate the variability of the C-OMT activity between different plants harvested at the same time. We see that it is relatively reduced.

The mutant corn line bm3 was used for the comparison. It was interesting to validate the protocol for measuring C-OMT activity, used for the first time on corn, using the mutant bm3, which exhibits zero C-OMT activity. Three bm3 plants were analyzed, and we observe a very low activity level corresponding to the background noise. The crude protein extracts from bm3 plants therefore constitute excellent negative controls in this work.

3 - C-OMT activity in transgenic populations

The results relating to the analysis of 32 different transformation events are summarized in FIG. 7. Since the analysis was carried out in certain cases on two batches of seeds of the same transformation event. Three plants from the same batch were analyzed in order to establish a standard deviation. Each crude extract was used for two activity determinations.

A total of 20 events with the ADH-ASOMT promoter (pProsper and pProsper-Bar) were analyzed. A significant reduction in COMT activity, of the order of 70%, was obtained for certain events.

Example 6: Histological analysis

A. Materials and Methods

Maϋle staining: The sections are incubated in a 1% (m / v) KMnθ ₄ solution for 5 minutes, rinsed in distilled water, discolored with a 15N HCl solution for 3 minutes, rinsed in distilled water, and incubated in a solution of NaHCO ₃ to 5% (m / v).

Phloroglucinol staining: The sections are immersed in a solution of phloroglucinol in hydrochloric solution (Prolabo). The photographs are taken with a camera. B. Results

The results are represented in FIG. 8, which represents photon microscopy pictures of cribovascular bundles of adult leaves of corn, respectively of the line I ₂ of control corn and of the line of corn 225, which was transformed with a antigen polynucleotide inhibiting methyltransferase COMT.

In FIG. 8, it can be seen that the coloration is less intense in the sclerenchyma and in the xylem of line 225 than in the sclerenchyma and the xylem of line I ₂ control. This reflects a decrease in S units in the lignins of the transgenic variety of corn 225.

EXAMPLE 7 Plant Digestibility Test by Assessment of Enzyme Solubility

A. Materials and Methods

This method makes it possible to estimate a potential digestibility by the activity of cellulolytic enzymes on the plant wall. The samples are placed in sealed bags and immersed in the enzyme solutions. The reactions take place in a DAIZY incubator which can contain four 2-liter bottles, shaken by rotation, maintained at 40 ° C and which can contain from 20 to 80 sachets. The lyophilized powder samples are incubated overnight at 70 ° C in a crystallizer. Then, 0.5 g of powder (initial mass) is placed in a sachet (F57, Ankom) which is closed by welding, incubated for 24 h at 70 ° C and then weighed after cooling (P1). The sachet is then incubated in a pepsin solution (Merck) at 20 g / L of 0.1 N HCL, at 40 ° C for 24 h, then at 80 ° C for Vz hour. The whole is then rinsed with water at 35 ° C and wrung. This step is repeated once. The sachet is then incubated in a cellulase solution (Onozuka R10 Yakult, Biochemical Co, Ltd.) At 1 g / L with amyloglucosidase (Merck) at 0.1 g / L in a sodium acetate / acetic acid buffer (2.95 mL of 96% acetic acid and 6.8 g of sodium acetate in 1L of demineralized water), at 40 ° C for 24 h, then rinsed and wrung twice, before being dried at the oven at 70 ° C for 24 hours. The bag is weighed (P2). The solubility (in% of the dry mass) is equal to (P1-P2) / (initial mass x 100).

B. Results The compared digestibility results are presented in the

Table 1 below.

Table 1: Digestibility index of corn plants

In Table 1, the index ndf represents the percentage of non-digestible fraction measured from lyophilized powder of whole plants harvested at the flowering stage.

In Table 1, the dndf index represents the digestibility index calculated from the value of ndf (neutral detergent fiber) and dms (enzymatic solubility Aufrère) according to the formula of Struik (1985).

The results of Table 1 show the higher digestibility of the two transgenic corn lines 225 (1) and 225 (2) which were transformed with an antisense construct according to the invention, compared to the control “WT” corn variety, the corn line I ₂ .

EXAMPLE 8 Evaluation of the Content, Composition and Structure of Transgenic Corn Lignin Subexpressing COMT: Evaluation by CPG-SM of the Monomeric Products Derived from Their Thioacidolysis

11 Study of lignins: content and structure

Three corn lines, one control (WT) and two COMT antisense lines from the same transformation event with residual activity 40% (3.2 and 1.2) harvested at the flowering stage, freeze-dried and crushed were analyzed.

The samples were extracted with an acetone / water mixture (5/1) in order to remove most of the soluble compounds (pigments, lipids, etc.) by a relatively rapid protocol. The results are presented in Table 2: the "residue" of the extraction roughly represents the percentage of walls in the sample.

Table 2: Results of the extraction of the control corn (WT) and transgenic samples

The fairly high extractable content is due to the presence of the leaves.

The observed values are quite similar for the 3 lines.

The Klason lignin assay was carried out on the extracted samples. It is expressed as a percentage by weight of these samples (Table 3).

Table 3: Klason lignin content of the corn samples (stems + leaves) extracted. Results expressed in% by weight of the walls extracted

The lignin contents are lower than in the case of stems harvested at maturity: the samples extracted come from (leaves + stems) harvested at the flowering stage. The two lines transgenic have a lower lignin content (25 to 30% less) than the control line.

The extractables content being similar for the 3 lines (Table 2), the difference between WT and antisense is maintained when the calculation is performed on the non-extracted sample

In order to assess the impact of transformation on the structure of lignins, whole plants (leaves + stems) were subjected to thioacidolysis (Lapierre et al., Plant Physiol 119, 153-163, 1999), followed by CPG-SM analysis of their trimethylsilylated derivatives (Satum Varian, electronic impact, 45-650 m / z). The relative frequency of p-hydroxyphenyl H, guaiacyl G, syringyl S and 5-hydroxyguaiacyl 5- OH G units engaged only in -0-4 bonds was determined on the chromatograms reconstructed on the most specific ions of the derivatives they generate (Figure 2). These ions, corresponding to the base peak of the mass spectrum, make it possible to unequivocally evaluate the proportions of the H / G / S / 5-OHG units linked in -0-4.

The results reported in Table 4 and in Figure 3 unequivocally reveal the efficiency of the transformation aimed at under-expressing the COMT activity. The transgenic lines 3.2 and 1.2 present a collapse in the frequency of the S units (divided by 2) and especially the appearance in their lignins of 5 OH-G units in substantial quantity (multiplied by a factor between 10 and 20). The effect is slightly more marked in the case of line 1.2. The differences observed between Tables 3 and 4 for the controls are due to the differences in organs analyzed, harvest stage and line. Table 4. Structure of corn lignins (stems + leaves harvested at flowering) from control lines and under-expressing COMT: Relative frequency of H, G, S and 5-OHG units linked in -0-4

21 Study of the cinnamic p-OH esters linked to the walls of extracted corn

The p-hydroxycinnamic esters bound to the walls were released by alkaline hydrolysis (1 M NaOH, 2.5 h, 40 ° C, 20 mg of sample extracted treated with 2 ml of sodium hydroxide, in the presence of 0.2 mg internal standard ethylvanillin, with stirring magnetic and under N ₂ ). The hydrolyzate was acidified with 2 M HCl (1.5 ml), ultrafiltered through Millex H25 NS (Millipore). The phenolic compounds were extracted by solid phase extraction on Sep-Pak C1.8 (Classic, Waters Millipore), according to a protocol adapted from Beveridge et al. (2000, Food Res. Internat., 33, 775-783). The Sep-pak cartridge was packaged with 2 * 5 ml MeOH, 2 * 5 ml water (milliQ), 2 * 5 ml formate buffer pH 3, before passage of the acidified hydrolyzate, followed by washing with 3 ml formate buffer. The elution of the phenolic compounds retained in full on the cartridge (we have verified it) was carried out with 1 ml CH ₃ CN. The collected sample was diluted v / v with the HPLC solvent before being ultrafiltered again and analyzed by reverse phase HPLC (20 μl injected). An identical treatment was carried out on a standard reference mixture containing equiponderal quantities of p-coumaric acid (PC), ferulic acid (Fe) and ethylvanillin (EtV), to carry out the calibration. HPLC analysis was carried out on a Lichrocart RP18 Merck column (5 μm, 250 * 4 mm), used under isocratic conditions, with elution by the water / CH ₃ CN / CH ₃ COOH mixture (78/20/2, v / v) at a flow rate of 1.3 ml / min and with detection at the column outlet at 280 nm. The results are reported in Table 5 and illustrated in Figure 4.

Table 5: Quantity of p-coumaric (PC) and ferulic (FE) acids released by mild alkaline hydrolysis (1M NaOH, 2.5h, 40 ° C) of extracted corn samples (stems + leaves). Results expressed in% by weight of the walls extracted

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Claims

1. Use of a polynucleotide modulating the synthesis of corn methyltransferase COMT coded by the nucleotide sequence SEQ ID No. 1, in a process for obtaining corn plants with improved digestibility characteristics having a ratio between the units S and G of modified lignin and / or having a modified lignin content remaining compatible with the normal development of the plant.

2. Use according to claim 1, characterized in that said polynucleotide is an antisense polynucleotide inhibiting the synthesis of COMT methyltransferase from corn.

3. Use according to claim 2, characterized in that the antisense polynucleotide comprises the nucleotide sequence SEQ ID

# 2.

4. Use according to claim 1, characterized in that said polynucleotide codes for methyltransferase COMT from maize, and causes a higher production of this enzyme in the plant.

5. Use according to claim 4, characterized in that the polynucleotide coding for methyltransferase COMT comprises the nucleotide sequence SEQ ID No. 1.

6. Method for obtaining maize plants having improved digestibility characteristics, having a ratio between the units S and G of the modified lignin and / or whose lignin content is modified and compatible with the normal development of the plant, said method comprising a step of transforming a corn host cell with a polynucleotide chosen from the following polynucleotides: a) an antisense polynucleotide specifically inhibiting the expression of the gene coding for COMT methyltransferase corn; b) a polynucleotide encoding COMT methyltransferase from corn.

7. Method according to claim 6, characterized in that it comprises the following steps: a) transforming a corn host cell with an antisense polynucleotide specifically inhibiting the translation of the gene coding for corn COMT methyltransferase .; b) regenerating an entire plant from the recombinant host cell obtained in step a);

8. Method according to claim 7, characterized in that the antisense polynucleotide comprises the nucleotide sequence SEQ ID

# 2.

9. Method according to claim 6, characterized in that it comprises the following steps: a) transforming a corn host cell with a polynucleotide encoding COMT methyltransferase corn; b) regenerating an entire plant from the recombinant host cell obtained in step a);

10. The method of claim 9, characterized in that the polynucleotide encoding COMT methyltransferase from corn comprises the nucleotide sequence SEQ ID No. 1.

11. Method according to one of claims 6 to 10, characterized in that it comprises the following additional step: c) selecting the corn plants having integrated into their genome at least one copy of the antisense polynucleotide or at least one copy of the polynucleotide encoding COMT methyltransferase from corn.

12. Method according to one of claims 6 to 11, characterized in that the antisense polynucleotide or the polynucleotide encoding corn methyltransferase COMT is integrated in an expression cassette comprising a nucleotide sequence regulating the expression of said polynucleotide in cells corn.

13. Method according to one of claims 6 to 12, characterized in that the antisense polynucleotide or the polynucleotide encoding COMT methyltransferase from corn is artificially inserted into a Agrobacterium tumefaciens cell.

14. Method according to one of claims 6 to 13, characterized in that in step a), the corn host cell is also transformed with at least one second polynucleotide, specifically modulating the expression of an enzyme involved in the lignin biosynthesis pathway, other than corn COMT methyltransferase.

15. The method of claim 14, characterized in that the second polynucleotide is chosen from: a) an antisense polynucleotide specifically inhibiting the expression of the gene encoding the enzyme involved in the lignin biosynthesis pathway, other than the COMT methyltransferase But ; b) a polynucleotide encoding the enzyme involved in the lignin biosynthesis pathway, other than corn COMT methyltransferase.

16. The method of claim 15, characterized in that the enzyme involved in the lignin biosynthesis pathway codes for an enzyme other than COMT methyltransferase from corn.

17. Method according to one of claims 6 to 16, characterized in that it comprises the following additional steps: d) crossing between them of two transformed plants as obtained in step b) or in step c ) with a second corn plant; e) selection of the plants homozygous for the transgene or the transgenes.

18. Method for obtaining a transformed plant according to one of claims 11 to 16, characterized in that it comprises the following additional steps: f) crossing of a transformed corn plant obtained in step c) with a second corn plant; g) selection of the corn plants resulting from the crossing of step f) and having preserved the transgene.

19. Corn cells capable of being obtained by the method according to one of claims 6 to 18.

20. A transformed corn plant capable of being obtained from the cells according to claim 19.

21. Part of a corn plant transformed according to claim 20, in particular, root, seed, stem, leaf or flower.

22. DNA construct comprising a polynucleotide modulating the synthesis of maize methyltransferase COMT, chosen from the nucleotide sequences SEQ ID No. 1 or SEQ ID No. 2 or a fragment thereof, under the control of linked regulatory sequences operatively to the DNA sequence to be transcribed.

23. DNA construct according to claim 22, characterized in that it also comprises a second polynucleotide coding for an enzyme involved in the biosynthesis of lignins, other than methyltransferase COMT from maize.

24. Recombinant expression vector comprising a polynucleotide chosen from: a) an antisense polynucleotide specifically inhibiting the expression of the gene encoding COMT methyltransferase from corn; b) a polynucleotide coding for methyltransferase COMT; said polynucleotide being placed under the control of a functional regulatory sequence in corn.

25. Recombinant expression vector according to claim 24, characterized in that it comprises a second polynucleotide, said polynucleotide being chosen from: a) an antisense polynucleotide specifically inhibiting the expression of the gene encoding the enzyme involved in the lignin biosynthesis pathway, other than COMT methyltransferase from corn; b) a polynucleotide encoding the enzyme involved in the lignin biosynthesis pathway, other than corn COMT methyltransferase.

26. Corn host cell or an Agrobacterium tumefaciens cell transformed with: a) either an antisense polynucleotide inhibiting the synthesis of methyltransferase COMT in a corn cell; b) or a polynucleotide coding for methyltransferase COMT, said polynucleotide (a) or said polynucleotide (b) being preferably placed under the control of a functional regulatory sequence in a corn cell, for example in an expression vector according to one of claims 24 or 25.

27. Transformation product obtained from a corn plant transformed according to claim 20, from a part of a corn plant according to claim 21, or from a corn cell transformed according to one of Claims 19 or 26, characterized in that it has improved digestibility characteristics and that it has a modified ratio between the S units and the G units of lignin and a modified lignin content.

28. Transformation product according to claim 27, characterized in that the ratio between the units S and the units G is less than 40/60.

29. Transformation product according to one of claims 27 and 28, characterized in that it is fodder.

30. Purified lignin obtained from a transformed corn plant according to claim 20, from a part of a corn plant according to claim 21, or from a corn cell according to one of claims 19 or 26 , characterized in that it has in particular a modified relationship between the S units and the G units.

31. Purified lignin according to claim 30, characterized in that the ratio between the units S and the units G is less than 40/60.