Means and methods for the production of woody plants with altered lignin composition

Abstract

The present invention provides chimeric genes and constructs which can be used to alter the lignin composition of woody plants and increase the saccharification and pulping efficiency of said plants. Particularly the lignin composition is modified by the incorporation of scopoletin.

Description

[0001] MEANS AND METHODS FOR THE PRODUCTION OF WOODY PLANTS WITH ALTERED LIGNIN COMPOSITION

[0002] Field of the invention

[0003] The present invention relates to the field of plant molecular biology, more particularly to the field of agriculture, even more particularly to the field of improving the saccharification and pulping efficiency of plants, particularly woody plants. The present invention provides chimeric genes and constructs which can be used to alter the lignin composition of woody plants and increase the saccharification and pulping efficiency of said plants.

[0004] Introduction to the invention

[0005] With the depletion of fossil resources, the need for a renewable and climate-neutral feedstock for the production of biobased products is a critical societal challenge (Vanholme et al., 2013a). Non-edible lignocellulosic biomass is the most abundant renewable carbon source on earth, and therefore a promising candidate. It consists primarily of carbohydrates (cellulose and hemicellulose) and an aromatic fraction (lignin). However, current lignocellulosic feedstock suffers from a relatively low processing efficiency, mainly because of the presence of the recalcitrant lignin polymer, even after pretreatment steps (Chen and Dixon, 2007; Yuan et al., 2021). To overcome this problem, biomass crops can be engineered to make either less lignin, or lignin with an altered composition (Vanholme et al., 2013b; Mottiar et al., 2016; Oyarce et al., 2019). Reducing the amount of lignin often results in a biomass yield penalty (Bonawitz and Chapple, 2013; Van Acker et al., 2014; De Meester et al., 2022a). Hence, new strategies to alter lignin composition are being explored (Wilkerson et al., 2014; Oyarce et al., 2019; Hoengenaert et al., 2022). In dicots, such as Arabidopsis and poplar, lignin is mainly made from the oxidative coupling of the monolignols p-coumaryl, coniferyl and sinapyl alcohol, that give rise to the p- hydroxyphenyl (H), guaiacyl (G) and syringyl (S) units of the lignin polymer, respectively. In addition, many monomers have been found to be incorporated in the lignin polymer, albeit often at lower levels (del Rio et al., 2020). A more recent strategy to alter the composition of the lignin polymer is to genetically engineer the plant such that it biosynthesizes a compound that could act as an alternative monomer; after its biosynthesis in the cytoplasm, this molecule crosses the plasma membrane to enter the cell wall where it is oxidized to a radical that than couples in the lignin polymer, in the same way as a normal lignin monomer does (Wilkerson et al., 2014). In the present invention we selected scopoletin as a candidate alternative monomer for lignin engineering. Upon incorporation of scopoletin via its O-4 function into the lignin polymer, a chemically labile bound, p-aryl ether with a conjugated carbonyl function, is introduced (see Figure 1). The presence of this carbonyl functionality facilitates the delocalization of electrons over a larger part of the molecule, a characteristic that has previously proven to strongly reduce the hydrolysis temperature necessary to break aryl ethers in an alkaline setting (Gierer and Ljunggren, 1979; Criss et al., 1998).

[0006] Scopoletin is biosynthesized from feruloyl-CoA, via FERULOYL-CoA 6'-HYDROXYLASE 1 or 2 (F6'H1 or F6'H2) and COUMARIN SYNTHASE (COSY), in Arabidopsis thaliana (Arabidopsis) (Kai et al., 2008; Vanholme et al., 2019). Introducing scopoletin into the lignin polymer has already proven to work in Arabidopsis resulting in scopoletin-overproducing lines with improved conversion of cellulose to glucose by 40% compared to WT, after alkaline pretreatment (Hoengenaert et al., 2022). Here we tried to translate this strategy to poplar trees but we surprisingly found that a multi-cistronic construct as used in transgenic Arabidopsis did lead to toxic effects. However, by reversing the order of the biosynthetic enzymes in the multi-cistronic construct we surprisingly obtained viable poplar plants with an increased saccharification efficiency.

[0007] Figures

[0008] Figure 1: Biosynthesis and incorporation of scopoletin into the lignin polymer. Scopoletin is synthesized through the phenylpropanoid pathway, involving enzymes F6'H1 (FERULOYL-CoA 6'-HYDROXYLASE 1) and COSY (COUMARIN SYNTHASE). The hypothesis suggests that the presence of a conjugated carbonyl in the scopoletin monomer makes the 8-0-4 bond between scopoletin and the A unit in the engineered lignin polymer more susceptible to cleavage under alkaline conditions, regardless of substitution at the 8- position. The arrow (in the fourth structure) highlights the bond with increased reactivity. It is important to note that alkaline conditions can also hydrolyze the cyclic ester (lactone), cleaving the bond marked with an asterisk (*), resulting in heightened hydrophilicity. This alteration makes the polymer more easily extractable into aqueous solvents. Additionally, it is worth mentioning that the numbering of scopoletin, depicted as a lignin monomer, adheres to standard lignin conventions rather than formal coumarin numbering (Hoengenaert et al., 2022).

[0009] Figure 2: Height of SC0P1, SC0P2 and SC0P3 transgenic poplars grown in greenhouse conditions. A: Height of poplars in batch 1 with one plant per independent line over a period of 23 weeks. B: Height of poplars in batch 2 with five repeats per independent line over a period of 23 weeks. C: Final height of poplars in batch 1 at day of harvest. D: Final height of poplars in batch 2 at day of harvest. Batch 1: 16, 13 and 1 independent line(s) for SC0P1, SC0P2 and SC0P3 transgenic poplars, respectively; n=l for all lines (A). Batch 2: 9, 9 and 1 independent line(s) for SC0P1, SC0P2 and SC0P3 transgenic poplars, respectively; n=5 for most lines (B and C). ** P<0.01 and **** P<0.0001 (One-Way ANOVA with Dunnett's post hoc). Figure 3: Scopoletin, scopoline and ferulic acid hexose accumulation in xylem of SCOP1, SCOP2 and SCOP3 transgenic lines. Peak intensity of scopoletin, its glucoside scopolin and ferulic acid hexose for each independent line for the three different constructs. Each independent line contains a single observation, as no biological repeats were grown in batch 1.

[0010] Figure 4: Lignin content in SCOP1, SCOP2 and SCOP3 transgenic poplars and WT. Lignin content from a selection of poplars from the first batch was determined using Klason assay and is expressed as a percentage of the CWR. For statistical analysis, the independent lines associated with each construct are considered as biological replicates for that specific construct. n=3, 5, 5 and 1, for WT, SCOP1, SCOP2 and SCOP3, respectively. *** P<0.001 (Welch's t-test).

[0011] Figure 5: Endpoint measurement of saccharification assay on SCOP1, SCOP2 and SCOP3 transgenic poplars. Alkali pretreated wood powder was incubated with a cellulase mixture for 72 hours. A: SCOP1 transgenics (batch 1) have a significant increase of on average 30% in sugar release compared to WT. B: Multiple independent SCOP1 lines (batch 2) displayed a significant increase in sugar release up to 29% compared to WT. C: Two independent SCOP2 lines (batch2) displayed a significant increase in sugar release compared to WT. D: SCOP3 line (batch 2) shows no significant difference in sugar release compared to WT. Significant differences are indicated by * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001 (Welch's t-test). CWR: cell wall residue.

[0012] Detailed description of the invention

[0013] The present invention provides plants, preferably woody plants which have scopoletin incorporated into their lignin polymer. Introducing scopoletin into the lignin polymer has already proven to work in Arabidopsis resulting in scopoletin-overproducing lines with improved conversion of cellulose to glucose by 40% compared to WT, after alkaline pretreatment (Hoengenaert et al., 2022). Here we investigated the translation of this same strategy to poplar trees but we surprisingly found that the same multi- cistronic construct as used in transgenic Arabidopsis did lead to toxic effects in poplar. However, poplar transformants harboring a multi-cistronic construct with the order of the biosynthetic enzymes reversed vis-a-vis the Arabidopsis construct were viable and furthermore such poplar plants had an increased saccharification efficiency. Without limiting the invention to a particular mechanism or action we believe that one reason why the novel multi-cistronic chimeric construct (further outlined below in example 1 as the SCOP1 construct) leads to viable poplar plants is because less of the toxic 6-hydroxyferulic acid glucoside is produced with recombinant poplars harboring the SCOP1 construct as compared with recombinant poplars harboring SCOP2 or SCOP3 constructs. Accordingly in a first embodiment the invention provides a multi-cistronic chimeric expression construct comprising a plant expressible promoter operably coupled to a nucleotide sequence encoding a coumarin synthase enzyme, a nucleotide sequence encoding a 2A peptide, a nucleotide sequence encoding a feruloyl-CoA-6'-hydroxylase enzyme and a plant terminator sequence wherein the coumarin synthase enzyme in the multi-cistronic chimeric expression construct lacks its natural stop codon sequence.

[0014] Thus to make it more clear that the order of the genetic elements is important in the multi-cistronic chimeric expression construct (and is different from the prior art) the invention provides a multi-cistronic chimeric expression construct comprising a plant expressible promoter operably coupled to a nucleotide sequence encoding a coumarin synthase enzyme followed by a nucleotide sequence encoding a 2A peptide followed by a nucleotide sequence encoding a feruloyl-CoA-6'-hydroxylase enzyme followed by a plant terminator sequence and wherein the coumarin synthase enzyme in the multi-cistronic chimeric expression construct lacks its natural stop codon sequence.

[0015] In yet another embodiment the invention provides a multi-cistronic chimeric expression construct comprising a plant expressible promoter operably coupled to a nucleotide sequence encoding a coumarin synthase enzyme, a spacer sequence, a nucleotide sequence encoding a 2A peptide, a nucleotide sequence encoding a feruloyl-CoA-6'-hydroxylase enzyme and a plant terminator sequence wherein the coumarin synthase enzyme in the multi-cistronic chimeric expression construct lacks its natural stop codon sequence.

[0016] In a particular embodiment the coumarin synthase enzyme is depicted in SEQ ID NO: 2.

[0017] In a particular embodiment the spacer sequence in the multi-cistronic chimeric expression construct is a nucleotide sequence encoding for GSG.

[0018] In another particular embodiment the spacer sequence in the multi-cistronic chimeric expression construct is a nucleotide sequence depicted in SEQ ID NO: 24.

[0019] In another embodiment the plant expressible promoter present in the multi-cistronic chimeric expression construct is a plant promoter active in cells that make a secondary cell wall.

[0020] In yet another particular embodiment the plant expressible promoter present in the multi-cistronic chimeric expression construct is the cellulose synthase 8-B promoter.

[0021] In another particular embodiment the cellulose synthase 8-B promoter is depicted in SEQ ID NO: 1.

[0022] In yet another embodiment the feruloyl-CoA-6'-hydroxylase enzyme is the feruloyl-CoA-6'-hydroxylase- 1 enzyme or the feruloyl-CoA-6'-hydroxylase-2 enzyme.

[0023] In another specific embodiment the feruloyl-CoA-6'-hydroxylase enzyme is a feruloyl-CoA-6' hydroxylase- 1 enzyme depicted in SEQ ID NO: 9. Orthologous sequences of the feruloyl-CoA-6' hydroxylase-1 enzyme are depicted in SEQ ID NO: 10 to SEQ ID NO: 16.

[0024] In another particular embodiment the feruloyl-CoA-6'-hydroxylase enzyme is a feruloyl-CoA-6' hydroxylase-2 enzyme depicted in SEQ ID NO: 17 to 23.

[0025] In another particular embodiment the 2A peptide in the multi-cistronic chimeric expression construct is a T2A peptide.

[0026] In a particular embodiment the T2A peptide is depicted in SEQ ID NO: 25.

[0027] In a particular embodiment the invention provides a plant cel I, a plant seed or a plant comprising a multi- cistronic chimeric expression construct as described herein above.

[0028] In a particular embodiment plant cells, plant seed or plants are cereals and grasses.

[0029] In another particular embodiment the invention provides a woody plant cell, a woody plant seed or a woody plant comprising a multi-cistronic chimeric expression construct as described herein above.

[0030] In yet another embodiment the invention provides the use of a multi-cistronic chimeric expression construct as described herein above to increase the saccharification and pulping efficiency of plants, particularly woody plants.

[0031] In yet another embodiment the invention provides a method for producing a woody plant with increased saccharification and pulping potential as compared to a corresponding wild type woody plant, whereby the method comprises transforming a multi-cistronic chimeric expression construct as described herein.

[0032] By "operably linked" is intended a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence. Generally, operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame.

[0033] The term "ortholog" means a member of a group of (orthologous) polypeptide or genes in different species, which have evolved from a common ancestral polypeptide / gene by speciation and which essentially retain the same biological function.

[0034] The term "saccharification" refers to the process by which raw plant material is converted by hydrolysis of into its constituent sugar moieties.

[0035] The term "pulping" or "wood pulping" refers tot the process by which woody tissue is treated with chemicals to degrade and extract lignin from the wood. By "plant" is intended whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, propagules, embryos and progeny of the same. Plant cells can be differentiated or undifferentiated (e.g. callus, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells, pollen). Consequently, the invention also includes a plant cell, seed or progeny derived from the genetically modified plant as defined above. In a preferred embodiment a plant is a woody plant.

[0036] According to the invention, the woody plant is preferably:

[0037] (a) a hardwood selected from the group consisting of acacia, eucalyptus, hornbeam, beech, mahogany, walnut, oak, ash, willow, hickory, birch, chestnut, poplar, alder, maple, sycamore, ginkgo, palm trees, and sweet gum; or

[0038] (b) a conifer selected from the group consisting of cypress, Douglas fir, fir, sequoia, hemlock, cedar, juniper, larch, pine, redwood, spruce, and yew.

[0039] Yet a further aspect of the invention is a method for the manufacture of wood pulp, said method comprising preparing a genetically modified woody plant as defined above; obtaining wood from the said woody plant; and converting the said wood into wood pulp.

[0040] For the purposes of the invention, "transgenic", "transgene" or "recombinant" means with regard to, for example, a nucleic acid sequence, an expression cassette, gene construct or a vector comprising the nucleic acid sequence or an organism transformed with the nucleic acid sequences, expression cassettes or vectors according to the invention.

[0041] A transgenic plant for the purposes of the invention is thus understood as meaning, as above, that the nucleic acids used in the method of the invention are not present in, or originating from, the genome of said plant, or are present in the genome of said plant but not at their natural locus in the genome of said plant, it being possible for the nucleic acids to be expressed homologously or heterologously. However, as mentioned, transgenic also means that, while the nucleic acids according to the invention or used in the inventive method are at their natural position in the genome of a plant, the sequence has been modified with regard to the natural sequence, and / or that the regulatory sequences of the natural sequences have been modified. Transgenic is preferably understood as meaning the expression of the nucleic acids according to the invention at an unnatural locus in the genome, i.e. homologous or, heterologous expression of the nucleic acids takes place. Preferred transgenic plants are mentioned herein above.

[0042] "Multi-cistronic or polycistronic constructs": the co-expression of multiple genes at a desired ratio is highly attractive for a broad array of basic research and biomedical applications including cellular reprogramming, expression of multiple subunits of complex multimeric proteins, tagging of protein of interest for live cell imaging or cell sorting, and generation of efficient tools for fate mapping and genome editing. Strategies for multigene co-expression include introduction of multiple vectors, use of multiple promoters in a single vector, fusion proteins, proteolytic cleavage sites between genes, internal ribosome entry sites, and "self-cleaving" 2A peptides. 2A peptides are 18-22 amino-acid (aa)-long viral oligopeptides that mediate "cleavage" of polypeptides during translation in eukaryotic cells. The designation "2A" refers to a specific region of the viral genome and different viral 2As have generally been named after the virus they were derived from. The first discovered 2A was F2A (foot-and-mouth disease virus)18, after which E2A (equine rhinitis A virus), P2A (porcine teschovirus-1 2A), and T2A (thosea asigna virus 2A) were also identified.

[0043] A "chimeric gene" or "chimeric construct" is a recombinant nucleic acid sequence in which a promoter or regulatory nucleic acid sequence is operatively linked to, or associated with, a nucleic acid sequence that codes for an mRNA, such that the regulatory nucleic acid sequence is able to regulate transcription or expression of the associated nucleic acid coding sequence. The regulatory nucleic acid sequence of the chimeric gene is not normally operatively linked to the associated nucleic acid sequence as found in nature.

[0044] The term "terminator" encompasses a control sequence which is a DNA sequence at the end of a transcriptional unit which signals 3' processing and polyadenylation of a primary transcript and termination of transcription. The terminator can be derived from the natural gene, from a variety of other plant genes, or from T-DNA. The terminator to be added may be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene.

[0045] The term "expression" or "gene expression" means the transcription of a specific gene or specific genes or specific genetic construct. The term "expression" or "gene expression" in particular means the transcription of a gene or genes or genetic construct into structural RNA (rRNA, tRNA) or mRNA with or without subsequent translation of the latter into a protein. The process includes transcription of DNA and processing of the resulting mRNA product.

[0046] The term "introduction" or "transformation" as referred to herein encompass the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer. Plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a genetic construct of the present invention and a whole plant regenerated there from. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem). The polynucleotide may be transiently or stably introduced into a host cell and may be maintained non-integrated, for example, as a plasmid. Alternatively, it may be integrated into the host genome. The resulting transformed plant cell may then be used to regenerate a transformed plant in a manner known to persons skilled in the art.

[0047] The transfer of foreign genes into the genome of a plant is called transformation. Transformation of plant species is now a fairly routine technique. Advantageously, any of several transformation methods may be used to introduce the gene of interest into a suitable ancestor cell. The methods described for the transformation and regeneration of plants from plant tissues or plant cells may be utilized for transient or for stable transformation. Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant, particle gun bombardment, transformation using viruses or pollen and microprojection. Methods may be selected from the calcium / polyethylene glycol method for protoplasts (Krens, F.A. et al., (1982) Nature 296, 72-74; Negrutiu I et al. (1987) Plant Mol Biol 8: 363- 373); electroporation of protoplasts (Shillito R.D. et al. (1985) Bio / Technol 3, 1099-1 102); microinjection into plant material (Crossway A et al., (1986) Mol. Gen Genet 202: 179-185); DNA or RNA-coated particle bombardment (Klein TM et al., (1987) Nature 327: 70) infection with (non-integrative) viruses and the like. Transgenic plants, including transgenic crop plants, are preferably produced via Agrobacterium-mediated transformation. An advantageous transformation method is the transformation in planta. To this end, it is possible, for example, to allow the agrobacteria to act on plant seeds or to inoculate the plant meristem with agrobacteria. It has proved particularly expedient in accordance with the invention to allow a suspension of transformed agrobacteria to act on the intact plant or at least on the flower primordia. The plant is subsequently grown on until the seeds of the treated plant are obtained (Clough and Bent, Plant J. (1998) 16, 735-743). Methods for Agrobacterium-mediated transformation of rice include well known methods for rice transformation, such as those described in any of the following: European patent application EP1198985, Aldemita and Hodges (Planta 199: 612-617, 1996); Chan et al. (Plant Mol Biol 22 (3): 491 - 506, 1993), Hiei et al. (Plant J 6 (2): 271 -282, 1994), which disclosures are incorporated by reference herein as if fully set forth. In the case of corn transformation, the preferred method is as described in either Ishida et al. (Nat. Biotechnol 14(6): 745-50, 1996) or Frame et al. (Plant Physiol 129(1): 13-22, 2002), which disclosures are incorporated by reference herein as if fully set forth. Said methods are further described by way of example in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1 , Engineering and Utilization, eds. S.D. Kung and R. Wu, Academic Press (1993) 128-143 and in Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991) 205-225). The nucleic acids or the construct to be expressed is preferably cloned into a vector, which is suitable for transforming Agrobacterium tumefaciens, for example pBinl9 (Bevan et al (1984) Nucl. Acids Res. 12-8711). Agrobacteria transformed by such a vector can then be used in known manner for the transformation of plants, such as plants used as a model, like Arabidopsis (Arabidopsis thaliana is within the scope of the present invention not considered as a crop plant), or crop plants such as, by way of example, tobacco plants, for example by immersing bruised leaves or chopped leaves in an agrobacterial solution and then culturing them in suitable media. The transformation of plants by means of Agro bacterium tumefaciens is described, for example, by Hofgen and Willmitzer in Nucl. Acid Res. (1988) 16, 9877 or is known inter alia from F.F. White, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1 , Engineering and Utilization, eds. S.D. Kung and R. Wu, Academic Press, 1993, pp. 15-38.

[0048] In addition to the transformation of somatic cells, which then have to be regenerated into intact plants, it is also possible to transform the cells of plant meristems and in particular those cells which develop into gametes. In this case, the transformed gametes follow the natural plant development, giving rise to transgenic plants. Thus, for example, seeds of Arabidopsis are treated with agrobacteria and seeds are obtained from the developing plants of which a certain proportion is transformed and thus transgenic [Feldman, KA and Marks MD (1987). Mol Gen Genet 208:1 -9; Feldmann K (1992). In: C Koncz, N-H Chua and J Shell, eds, Methods in Arabidopsis Research. Word Scientific, Singapore, pp. 274-289], Alternative methods are based on the repeated removal of the inflorescences and incubation of the excision site in the center of the rosette with transformed agrobacteria, whereby transformed seeds can likewise be obtained at a later point in time (Chang (1994). Plant J. 5: 551 -558; Katavic (1994). Mol Gen Genet, 245: 363-370). However, an especially effective method is the vacuum infiltration method with its modifications such as the "floral dip" method. In the case of vacuum infiltration of Arabidopsis, intact plants under reduced pressure are treated with an agrobacterial suspension [Bechthold, N (1993). CR Acad Sci Paris Life Sci, 316: 1 194-1 199], while in the case of the "floral dip" method the developing floral tissue is incubated briefly with a surfactant-treated agrobacterial suspension [Clough, SJ and Bent AF (1998) The Plant J. 16, 735-743], A certain proportion of transgenic seeds are harvested in both cases, and these seeds can be distinguished from non-transgenic seeds by growing under the above-described selective conditions. In addition the stable transformation of plastids is of advantages because plastids are inherited maternally is most crops reducing or eliminating the risk of transgene flow through pollen. The transformation of the chloroplast genome is generally achieved by a process which has been schematically displayed in Klaus et al., 2004 [Nature Biotechnology 22 (2), 225-229], Briefly the sequences to be transformed are cloned together with a selectable marker gene between flanking sequences homologous to the chloroplast genome. These homologous flanking sequences direct site specific integration into the plastome. Plastidal transformation has been described for many different plant species and an overview is given in Bock (2001) Transgenic plastids in basic research and plant biotechnology. J Mol Biol. 2001 Sep 21; 312 (3):425-38 or Maliga, P (2003) Progress towards commercialization of plastid transformation technology. Trends Biotechnol. 21 , 20-28. Further biotechnological progress has recently been reported in form of marker free plastid transformants, which can be produced by a transient co-integrated maker gene (Klaus et al., 2004, Nature Biotechnology 22(2), 225-229).

[0049] The genetically modified plant cells can be regenerated via all methods with which the skilled worker is familiar. Suitable methods can be found in the abovementioned publications by S.D. Kung and R. Wu, Potrykus or Hofgen and Willmitzer.

[0050] Generally after transformation, plant cells or cell groupings are selected for the presence of one or more markers which are encoded by plant-expressible genes co-transferred with the gene of interest, following which the transformed material is regenerated into a whole plant. To select transformed plants, the plant material obtained in the transformation is, as a rule, subjected to selective conditions so that transformed plants can be distinguished from untransformed plants. For example, the seeds obtained in the above-described manner can be planted and, after an initial growing period, subjected to a suitable selection by spraying. A further possibility consists in growing the seeds, if appropriate after sterilization, on agar plates using a suitable selection agent so that only the transformed seeds can grow into plants. Alternatively, the transformed plants are screened for the presence of a selectable marker such as the ones described above.

[0051] The generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or Tl) transformed plant may be selfed and homozygous second-generation (or T2) transformants selected, and the T2 plants may then further be propagated through classical breeding techniques. The generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and nontransformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion). Examples l.lntroduction of the scopoletin biosynthetic pathway in poplar

[0052] To introduce the scopoletin biosynthetic pathway into the lignifying tissues of Populus tremula x P. alba (poplar), the coding sequences of F6'H1 and COSY originating from Arabidopsis were cloned under transcriptional control of the secondary cell wall-specific CELLULOSE SYNTHASE 8-B (CesA8-B) promoter from Populus trichocarpa. Three different constructs were generated: pCesA8B:COSY_F6'Hl (SCOP1) and pCesA8B:F6'Hl_COSY (SCOP2) were both made as a bicistronic construct linked by a GSG spacer and a T2A sequence. These constructs differ by the order of the F6'H1 and COSY genes. The order of F6'H and COSY genes in the SCOP2 construct is the same as in the construct successfully used by Hoengenaert et al (2022) in Arabidopsis. The third construct pCesA8B:F6'Hl (SCOP3) aimed at expressing F6'H alone. Because the reaction catalyzed by COSY is also catalyzed by light, we wanted to investigate with this latter construct whether solely F6'H is sufficient to boost scopoletin biosynthesis in poplar wood.

[0053] The three SCOP constructs were transferred into poplar via Agrobacterium-m ediated transformation. A total of 52 independent transgenic lines were generated: twenty-one lines with SCOP1, seventeen with SCOP2 and fourteen lines containing SCOP3. In general, poplars containing the SCOP3 construct exhibited a severely dwarfed phenotype ultimately resulting in the death of twelve out of the fourteen independent lines.

[0054] The resulting transgenic poplars were analyzed in two separate batches. In batch 1, one plant per independent line was transferred from in vitro to soil to perform preliminary experiments to select the most interesting lines that would be further analyzed with the proper number of repeats per independent line. In batch 2, a selection of poplars was included based on findings from the first batch and planted in soil with five replicates per independent line. Additionally, four and five independent lines, that were not included in batch 1, were introduced in batch 2 for SCOP1 and SCOP3, respectively, with five repeats per independent line.

[0055] 2.Phenotypic analysis of transgenic poplar trees

[0056] For both batch 1 and 2, poplars grew in soil in greenhouse conditions and their height was measured every week over a period of 23 weeks (Figure 2 and Table SI). On average, SCOP2 transgenic poplars showed a reduced growth as compared to WT. Considering that a similar construct, namely one with F6'H1 positioned in front of COSY separated by a T2A sequence, was used in Arabidopsis without negative effect on growth, this outcome was rather unexpected (Hoengenaert et al., 2022). In contrast, most lines with the SCOP1 construct (i.e., with COSY positioned in front of F6'H1), had a growth rate similar to the wild type (WT) poplars. The two surviving lines with the SCOP3 construct (one in each of the two batches) grew similar as WT.

[0057] After 15 weeks growth on soil, the overall phenotype of the trees was evaluated for all SCOP lines. All SCOP3 independent lines generated for batch 1, except for line 8, died upon transfer to soil. For SCOP2 lines the most extreme phenotypes did survive but had a height reduction of up to 91% compared to WT, whereas for SCOP1 lines the most extreme height reduction was 33% compared to WT (Figure 1, line 1- 15). Therefore, both the presence of COSY, and the order of COSY and F6'H1 on the construct appear to be crucial for survival and growth of these transgenic poplars. It is clear from Figure 2 that the growth of SCOP1 plants is better than that of SCOP2 plants. These data indicate that the product of the F6'H1 reaction, or derivatives thereof, are toxic to the tissue in which they are produced.

[0058] 3.Metabolic analysis of transgenic poplar trees

[0059] To investigate the impact of F6'H1 and COSY expression in poplar lignifying cells on the phenolic metabolism, methanol-soluble metabolites were extracted from xylem of poplars grown in batch 1 and subjected to analysis using UHPLC-MS. In general, SCOP1 transgenic poplars contained more scopoletin and scopolin (scopoletin glucoside) (Figure 3) than SCOP2 and SCOP3 transgenic poplars, highlighting that the position of the genes relative to the T2A sequence as well as the presence of COSY play a crucial role in the production of scopoletin. To explain the abnormal phenotypes in SCOP2 and SCOP3 plants, we hypothesized that SCOP2 and SCOP3 lines produce products that are toxic to the cells. We found that the levels of the glucoside of 6-hydroxyferulic acid, the latter being the de-esterification product of 6- hydroxyferuloyl CoA, is higher in SCOP2 transgenic poplars compared to WT and SCOP1 and SCOP3 poplars (see Figure 3). One would anticipate the production of 6-hydroxyferulic acid in SCOP3 poplars as well. However, SCOP3-1 failed to produce 6-hydroxyferulic acid, scopoletin, or scopolin, therefore the presence of the SCOP3 construct was analyzed using PCR. The results indicated the absence of the construct in SCOP3-1, providing an explanation for the absence of 6-hydroxyferulic acid glucoside and the absence of an abnormal plant phenotype in this line. Based on the height measurements and the scopoletin and scopolin production levels, a selection of the most interesting lines of batch 1 was made for further analysis. We therefore selected a couple of lines with relatively high levels of scopoletin and / or scopolin, that either grew like WT (SCOP1-7, SCOP1-15, SCOP1-20, SCOP2-14 and SCOP2-22) or that remained smaller than WT (SCOP1-2 SCOP1-5, SCOP2-7, SCOP2-8 and SCOP2-12). The one surviving line with the SCOP3 construct, SCOP3-1, was included in the selection of batch 1, even though no scopoletin and only marginal levels of scopolin were detected, this was the only line of this construct that survived in this batch. 4.Lignin analysis of transgenic poplar trees

[0060] To determine the lignin content, the above-mentioned selection of poplars from the first batch were analyzed by the Klason method. Briefly, cell wall residue (CWR) was treated with sulfuric acid to remove polysaccharides, after which acid-insoluble lignin was determined gravimetrically. Compared to WT, SCOP1 transgenics had significantly less lignin (on average a reduction of 7.6%; see Figure 4). On average, the lignin levels of SCOP2 transgenics did not show significant differences compared to WT. For SCOP3, lignin levels of only a single tree could be measured due to the lethal effects of that construct in the other SCOP3 lines, implying that no statistical tests could be performed. However, the lignin amount in the single SCOP3 tree fell within the range of WT observations, hinting no effect on lignin amount for that line, which is also in agreement with the absence of notable increases in scopoletin levels.

[0061] 5.Saccharification assay of transgenic poplar trees

[0062] Introduction of scopoletin into the lignin polymer of Arabidopsis has previously resulted in a 40% higher saccharification efficiency compared to WT upon alkaline pretreatment. To test the saccharification potential of SCOP poplars, a saccharification assay was performed on a subset of SCOP poplars originating from the two batches, the selected lines of batch 1 (1-2, 1-5, 1-7, 1-15, 2-7, 2-8, 2-12, 2-14 and 2-22) and all the independent lines of batch 2 (1-2, 1-4, 1-5, 1-7, 1-8, 1-12, 1-15, 1-24, 1-25, 2-1, 2-5, 2-7, 2-8, 2-12, 2-14, 2-21, 2-22 and 3-8). After an alkaline pretreatment (62.5 mM NaOH for 3 hours at 90°C), poplar wood powder was incubated with a cellulase enzyme mixture for a period of 72 hours. In poplars grown in batch 1 (Figure 5-A), independent lines were used as repeats of the constructs. On average, SCOP1 transgenics displayed a 30% increase in sugar release compared to WT. On the other hand, saccharification yield of SCOP2 transgenics and the single SCOP3 transgenic was not different from that of WT. In poplars grown in batch 2, seven out of nine analyzed SCOP 1 lines (Figure 5-B) showed a significant increase in sugar release compared to wild type with a maximum increase of 29% for line 1-2 compared to WT. When we take the growth phenotype into account, only line 1-5, displaying a significant increase in sugar release, had reduced growth. For SCOP2 transgenics from batch 2, only two out of the seven analyzed lines (line 5 and 21) showed increased sugar release compared to WT (Figure 5-C) with a maximum increase of 28% for line 5 compared to WT. However, both lines displayed a dwarfed phenotype which leaves no promising lines for industrial purposes with SCOP2 lines. Additionally, no pretreatment and an acid pretreatment (1 M HCI for 2 hours at 80°C) were also tested for SCOP1 poplars of batch 2, but the saccharification of the wood from these transgenic lines was not different compared to WT (data not shown). In conclusion, SCOP 1 lines produce significantly most scopoletin and scopolin, and less 6-hydroxyferulic acid hexose, while normal growth is maintained in most lines. SCOP2 lines produce less scopoletin and scopolin than SCOP1 lines, and accumulate more ferulic acid hexose, derived from the intermediate 6- hydroxy feruloyl-CoA. The SCOP2 lines that had improved saccharification were all dwarfed, whereas all but one SCOP1 line that had improved saccharification had normal growth. Plants that had normal growth and improved saccharification were only obtained from the SCOP1 construct where COSY is positioned upstream of F6'H. We conclude that both the presence of COSTin the construct, and the order of the F6'H and COSY coding sequences in the bi-cistronic construct are important in determining the scopoletin / scopolin levels and the levels of intermediates of the pathway that are converted into toxic metabolites.

[0063] 6.Field trial with the SCOP1 poplar lines

[0064] Three SCOP1 lines (SCOP1-2, SCOP1-7, and SCOP1-8) previously demonstrated promising results in greenhouse settings, including a respective increase of 29, 22 and 15% in saccharification efficiency compared to wild-type (WT) control. Therefore, a field trial was started on May 29, 2024, in Wetteren, Belgium, where we aimed to evaluate their performance under outdoor conditions. The field trial, designed with a randomized complete block design, comprised five blocks with eight replicates per genotype per block, resulting in a total of 160 poplars). Border plants were included to minimize edge effects but were not included in the experimental analyses.

[0065] Height measurements were recorded approximately every two weeks and plotted over time. While all lines exhibited similar height until late August, SCOP1-2 and SCOP1-7 subsequently deviated, showing a significant reduction in height compared to WT. However, no significant differences of height of SCOP1- 8 compared to WT could be observed. Diameter measurements were taken once in winter after growth cessation. Diameter of SCOP1-2 and SCOP1-7 is significantly reduced compared to WT. SCOP1-8, however, did not show significant differences compared to WT. Next, all lines were monitored for insect and rust infections, but no significant differences compared to WT were observed.

[0066] NMR analysis of greenhouse-grown poplars revealed significantly higher scopoletin levels in the lignin of all lines, but most in lines SCOP1-2 and SCOP 1-7 (2% and 2.2%, respectively), and relatively less in line SCOP1-8 (1.26%). The height and diameter differences observed in SCOP1-2 and SCOP1-7 may therefore be attributed to either the higher incorporation of scopoletin in the lignin polymer, or the higher levels of scopolin (scopoletin glucoside) or the higher levels of the 6-hydroxyferuloyl-CoA (the intermediate in the scopoletin biosynthetic pathway) or derivatives thereof. Notably, although SCOP1-8 also incorporates scopoletin in lignin and has improved saccharification, it has normal growth in the field. 7.Sequences

[0067] SEQ ID NO: 1 pCesA8B promotor sequence

[0068] GACACCGAATGCTTTACACAACCCACAATCATATAAAAAATTAATTCAGACATCAAGCAAGCCATAGGACATGCTAG

[0069] CTAAAAATATAGACAGAAAAGGTTGTGTATACACGCAAAAACATACTCATCGAGGACAATACTTGCATCAGCAAGAC

[0070] ATTAATTTGATAGTCGCATCCTTTGACAGAAGTTCTTGAAAGTCATCACAATGAATAAAAGTTGTCAATCCACTAGTA

[0071] AAGAATTCTGAAACAAGAGCCTGTAAAATTTTTAAAATATATTCATTAGCAAAATGGAAAGTAACTAGGAAAAAAAT

[0072] ATTGAATTAAGTTTCTTTGGCTATGTTATTCCCTCTTTCAAGAGATTTTGTTTTTGTTATTGTTGATTTTCTTCCGGCTT

[0073] TCTACTCTCCTTTTTCTCAGGAATCAAAGACCTTGGTTTTTCCAGATCAAGGACCCTAGCCAATTTGCCTATAATAGG

[0074] CCTCTAAAACCCGTTTCCCATGAAAGGAGAAAGCATAGGGAGAAAGAGAAAGTGAGCAAGCGTAGTGAGAAAAA

[0075] CAGAAAATTAGGAGAAAAAAAGAAGGAAGATGAGATAGAAAAGAGAAAGAAATAGACAGCAAAAAAGCACACA

[0076] GGGAAGAAAAAAGATAAAGAAAAGATAGAAAGAAAATCGAGATATAAAAGAATAAAAAGGAAAAAAGAAGTTTT

[0077] TTTAAGGTTGAGAAAAAAGTAAGGAAGAGAATCTTGAGATTTTGCAAGAAATAGGTTCCATTTGGACTACTCTGTG

[0078] GGTGGCTATGTTGCTGCTGTGTCCTGTGTCACATTTTCAATGTTCGGGGAGAAGGGGAACCTGATAATTTCTGGTG

[0079] GAAATGATAAGTTGATCAGTGAAAATGTGGGATTGCTCCAAATATCATGAAGCAGGGCAGACCGATGGTAACGAAG

[0080] ATGTTCTACGTCTTAACATTGATTTGAGCAAAAGGTCGATTTTTATTGTCTTTTGCTCGTGTTGGAATCCTTTCATCTC

[0081] ATACTTTCCATTTTGGCAGGTCAATTGGTTCTGTAAAACTCCAACTGATTCAGAAAACTTGTTGTTTGTGACGCAAA

[0082] AAAAGTAGTAGAGGTCAATTCTGTTTCTTAGATTAGAGCTACAACATCATAGACTCCTGGAGAGCATGCCTTGATTCT

[0083] GTTCAGGAGACGATAGTTTCCGGTTCGTTGAATGGCTTTGTTCACTTCTGGTCTAGCAATTTGCAAAAGAAGTTACA

[0084] AAACAAATGCATATTATGTAAATTTAACAAGAGATGGGTTCTATAGTCACTTATTTATGCCCATAATTTGTTCTGGGGT

[0085] TACTCTTTATAGTCTGATTCGAAGTTGCAAACTGCCGTTTCTGGTATTGCAATTATGTAGCCATAAACTGTTAATCCTG

[0086] TTCCTATTAGTGGACCAACAACCAGATATATGGGCTCAGCGTCGTAAAAGAGATCTCCATTCTACGTTTCTTCCTATTT

[0087] TTTCCGTTTCAGTGAGAGAATTACCCTGATACATTGATATGATGATTGATGATTATGGGAACCATGCCGATGTTAGAC

[0088] ACTAGACCATCTGGATCCTGCCAGTTTTCTGTTCACATGGCATCCCAGCCCAAGATCATGTGTTTATACTCACTAATGA

[0089] CTTGTATTGAAAGTTTGTTAGTTGAAGATGTGCTCTGCCCAACAGAAACCTTCCTTAAATTTCCCGCAAATTTTTCAA

[0090] AACTTGTCACTTACACCCCAAAAAATAGACGTTGCTTCTCACTTATGTTTCTCTGCAAAACACATGACACCAACTCCC CAACCGCCATACCCCACCAACCCACCACCCTCAACCTTCTCTTCGCCATTACAAAAATGTCAGTACCACCCTCTGAAA GACACCAACACACCCTAGCTTTGGTTAGGGTATTTGATATAAAAACAAGGCCAAAACAAAAGATTGGAAGGAAGC

[0091] AGAGGAAGACCCTCTTGAAAGAATTGAAGAATTGGAGTTGTAAAGAGCTGGTAAAGTGGTAATAAGCAAG

[0092] COSY sequences

[0093] SEQ ID NO: 2 Arabidopsis thaliana COSY sequence

[0094] MATLEITDIALVQPSHQPLSNDQTLSLSHLDNDNNLHVSFRYLRVYSSSSSTVAGESPSAVVSASLATALVHYYPLAGSLR

[0095] RSASDNRFELLCSAGQSVPLVNATVNCTLESVGYLDGPDPGFVERLVPDPTREEGMVNPCILQVTMFQCGGWVLGAS

[0096] IHHAICDGLGASLFFNAMAELARGATKISIEPVWDRERLLGPREKPWVGAPVRDFLSLDKDFDPYGQAIGDVKRDCFF

[0097] VTDDSLDQLKAQLLEKSGLNFTTFEALGAYIWRAKVRAAKTEEKENVKFVYSINIRRLM NPPLPKGYWGNGCVPMYAQ IKAGELIEQPIWKTAELIKQSKSNTSDEYVRSFIDFQELHHKDGINAGTGVTGFTDWRYLGHSTIDFGWGGPVTVLPLSN KLLGSMEPCFFLPYSTDAAAGSKKDSGFKVLVNLRESAM PEFKEAMDKFHKGEFALS

[0098] SEQ ID NO: 3 Arabidopsis lyrata: AL1G42300 (96.0% identity with SEQ ID NO: 2)

[0099] MATLEITEIALVQPSHQPLSNDQTLSLSHLDNDNNLHVSFRYLRVYSSSSTVAGKSPSEVVSASLATALVHYYPLAGSLRR

[0100] SATDNRFELYCAAGQSVPLVNASVNCTLESVGYLDGPDPGFVERLVPDPTREEGMVNPCILQVTMFQCGGWVLGAAI

[0101] HHAICDGLGASLFFNAMAELARGATKISIEPAWDRERLLGPREKPWVGAPVRDFLSLDKDFDPYGQAIGDVKRECFFV

[0102] TDESLDQLKAQLLEKSGVNFTTFEALGAYIWRAKVRAAKIEEKENVKFVYSINIRRLMNPPLPKGYWGNGCVPMYAQIK AGELIEQPIWKTAELIKQSKSNTSDEYVRSFIDFQELHYKDGINAGTGVTGFTDWRYLGHSTIDFGWGGPVTVLPLSNKL LGSMEPCFFLPYSTDAAAGSKKDSGFKVLVNLRESAMPEFKEAMEKFHKGEFSRS

[0103] SEQ ID NO: 4 Capsella rubella: Carub.0001s2711 (92.3% identity with SEQ ID NO: 2)

[0104] MATHEITETALVQPSHQPLSNDQTLSLSHLDNDNNLHVSFRYLRVYSSSSSSSTVAGKSPSSVVSASLATALVHYYPLAGS

[0105] LRRSASDNRFELFCTAGQSVPIVNASVNCTLESVAYLDGPDPGFVERLVPDPTREEGMVNPCILQVTTFQCGGWVLGA

[0106] SIHHAICDGLGASLFFNAMAELARGATKISIEPVWDRAQLLGPREKPWIGSPVRDFLSLVKDFDPYGLAIGDVKRECFFV TDESLDRFKAQLLEKSGLNFTTFEALGAYIWRAKVRAAKIGEEENVKYVYAINIRRLLNPPLPKGYWGNGCVPMYAQIK AGELIEQPIWKTAELIKQSKSNASDEYVRSFIDFQELHHKDGINAGSGVTGFTDWRYLGHSTIDFGWGGPVTVLPLSNK LLGSMEPCFFLPYSADAAAGSKDSGFKVLVNLRESAMPEFKEAMEKFHKGEIALS

[0107] SEQ ID NO: 5 Quercus lobata: QL01p001225 (72.0% identity with SEQ ID NO: 2)

[0108] MRKQVKPPLPAGYWGNGCVPMYAQLSAKELVEKPIWETAQLINKSKSRATDEYVRSFIDFQHLHYGDGITAGKGVSGF TDWRHLGHSTVDFGWGGPVTVLPLSRNLLGSVEPCFFLPYSSVSAGRKDGFKVLVNLRESAMPAFKEEMKEFGNHEF GLSRY

[0109] SEQ ID NO: 6 Durio zibethinus: Duzib235G0533 (63.5% identity with SEQ ID NO: 2)

[0110] MNVQVTETALIRPSTPPFTEDHALLLSHLDNDHSLNVTFRYLRAYVNSNTSDRNPFQVISSAISTALHHYYPLAGSLHRA SNGRYELFCQVDQSLPLVNASADCTLESVNYLDDPDMNSVEQLVPDPSPEETLVNPCILQLTVFKCGGFTLGAAIHNAL CDGLGATQFFCLAADLARGVDQVKFQPVWDRAALLGPRNPPKVEGPVRDFLSLEKGFNPYKQNIGHVVRECFYVEDE CLDQLKALLSEQSGLSLTTFEILGAYIWRAKVQASKIPGDETVKFSYLMNIRKLVKPPLPAGYWGNGCVAIYAKVSAKDLI EQPLWKTAELIKKSKSNSSDEYVRSFIDLQELHYEEGITAGKGVSGFTDWRHLGHSAVDFGWGGPVTVLPLSSNFLGSM EPCFFLPYSSSNTGKNKGFKVLVSLRESAMPAFREEMEKFSRKEFSRL

[0111] SEQ ID NO: 7 Populus trichocarpa: Potri.004G053500 (62.9% identity with SEQ ID NO: 2)

[0112] MEEIHIKETIPIRPSTPPFSQDHTLPLSHLDTDRNLNVTFRYLRVYVNTTTSNGGHPFNVIAAALSSALVHYYPLAATLRR GQVDDRLELFCTRDHLGVPLINATVNCTLEKLNYLDDSDPNFLDGLVPDPDQDYGLANPCVLQVTVFECGGWTLGAAI

[0113] HHGLCDGLGATQFFNVMAELARGVGRISANPVWDRARLLGPRDPPRAEGVVREFLGLEKGSEPYGQWGEVVRECF

[0114] PVKDEWLEKFKKVLFEKSGSSFTTFEALGAFIWRAKVKASGVPGDENVKFAYSINIRKLVKPPLPAGYWGNGCVPMYA QLCARELIEQPVWKTAELIKKSKINATDEYVRSFIDFQELHYGDGITAGNRVSGFTDWRHLGHSTVDFGWGGPVTVLPL SRKLLGSVVPCFFLPYSSANAGKKDGFKVLVTLQETHMPAFKKEMEKFSRQDFDLS

[0115] SEQ ID NO: 8 Eucalyptus grandis: Eucgr.D00550 (55.0% identity with SEQ ID NO: 2)

[0116] MQVQIHETKLIQPATPPLPDQEPVLPLSHLDTDPNLDITFRYLRVYVNADQTKRAHDTPYHVIARALSGALCYYYPLCGT LRPGDNDHHRLELHCVAGQGMPLVHATANCTLHAVKNLDDADSGFLEQLIPNPDPEESLANPCMLQVTVFECGGFSL GTAIHHTMCDGLGATLFFGAAAELARGAERVSIEPVWDRAVLLGPRSPARIEALVGDYLCEKGRNPYDDEENCKFGREF FHVKEEWLDRFKALMLEKCGQKFTTFEALGAFIWRAKVKVSDIPGDETVKFSYSTNIRKVLKPPLPVGYWGNGCVPIYV QLTARDLLEKPIWEVAELIRKSKRNASDEYVRSYIDFQEAHRREGVSAGKNVSGFTDWRHLGHSAVDFGWGGPANVL PLSRKLLGSNEACFFLPCSEVGVRDPSDGFRVLVNLKDRCLCSFKEEMEKLGKMEFGFA

[0117] F6'Hl-sequences

[0118] SEQ ID NO: 9 Arabidopsis thaliana

[0119] MAPTLLTTQFSNPAEVTDFWYKGNGVKGLSETGIKALPEQYIQPLEERLINKFVNETDEAIPVIDMSNPDEDRVAEAVC

[0120] DAAEKWGFFQVINHGVPLEVLDDVKAATHKFFNLPVEEKRKFTKENSLSTTVRFGTSFSPLAEQALEWKDYLSLFFVSE AEAEQFWPDICRNETLEYINKSKKMVRRLLEYLGKNLNVKELDETKESLFMGSIRVNLNYYPICPNPDLTVGVGRHSDV SSLTILLQDQIGGLHVRSLASGNWVHVPPVAGSFVINIGDAMQIMSNGLYKSVEHRVLANGYNNRISVPIFVNPKPESV IGPLPEVIANGEEPIYRDVLYSDYVKYFFRKAHDGKKTVDYAKI

[0121] SEQ ID NO: 10 Arabidopsis lyrate: AL3G25540 (96.4% identity with SEQ ID NO: 9)

[0122] MAPTLSTTQFSNPAEVTDFVVHKGNGVKGLSETGIKALPDQYIQPFEERLINKFVNETDEAIPVIDMSNPDENRVAEAV

[0123] CDAAEKWGFFQVINHGVPLEVLDDVKAATHRFFNLPVEEKCKFTKENSLSTTVRFGTSFSPLAEQALEWKDYLSLFFVSE

[0124] AEAEQFWPDICRNETLEYIDKSKKMVRKLLEYLGKNLNVKELDETKESLFMGSIRVNLNYYPICPNPDLTVGVGRHSDVS SLTILLQDQIGGLHVRSLASGNWVHVPPVPGSFVINIGDAMQILSNGRYKSVEHRVLANGNNNRISVPIFVNPKPESVI GPLPEVIANGEEPIYRDVLYSDYVKYFFRKAHDGKKTVDYAKI

[0125] SEQ ID NO: 11 Capsella rubella: Carub.0003sl307 (93.4% identity with SEQ ID NO: 9) MAPTLSTAQFSTPAEVTDFVVHRGNGVKGLSETGIKALPDQYIQPLEERLINKFVNETDEAIPVIDMSNPDEKKVAEAVC

[0126] DAAEKWGFFQVVNHGVPLEVLDNVKAATHRFFNLPVEEKSKFTKENSLSATVRFGTSFSPLAEKALEWKDYLSLFFVSD

[0127] AEAEQFWPDACRNETLEYIDKSKKMVRKLLEYLGKNLNVKELDETKESLFMGSIRVNLNYYPICPNPDLTVGVGRHSDV SSLTILLQDQIGGLHVRSLASGNWVHVPPVPGSFVINIGDAMQILSNGLYKSVEHRVLANGSNNRISVPIFVNPKPESVI GPLPEVIAKGEEPIYRDVVYSDYVKYFFRKAHDGKKTVDFAKI

[0128] SEQ ID NO: 12 Arabidopsis thaliana: AT1G55290 (77.4% identity with SEQ ID NO: 9)

[0129] MNQTLAAQFLTRDQVTNFVVHEGNGVKGLSETGIKVLPDQYIQPFEERLINFHVKEDSDESIPVIDISNLDEKSVSKAVC

[0130] DAAEEWGFFQVINHGVSMEVLENMKTATHRFFGLPVEEKRKFSREKSLSTNVRFGTSFSPHAEKALEWKDYLSLFFVSE

[0131] AEASQLWPDSCRSETLEYMNETKPLVKKLLRFLGENLNVKELDKTKESFFMGSTRINLNYYPICPNPELTVGVGRHSDVS SLTILLQDEIGGLHVRSLTTGRWVHVPPISGSLVINIGDAMQIMSNGRYKSVEHRVLANGSYNRISVPIFVSPKPESVIGPL LEVIENGEKPVYKDILYTDYVKHFFRKAHDGKKTIDFANI

[0132] SEQ ID NO: 13 Quercus lobata: QL01p008285 (67.9% identity with SEQ ID NO: 9)

[0133] MAPTIATPISESSNITDFVVTNGNGVKGISEMGLKTLPKQYIQPVEERITVSNILPQESIPIIDMSNWDEQKVSESICDAA

[0134] EKWGFFQIINHGVPIEVLENVKVATHRFFNLPAEEKRKFSKENSPSNSVRFGTSFSPEAEKALEWKDYLSLFYVSEDEVSA

[0135] LWPSACKDQVLEYMRGSELVIQRLLEALMKRINVKEIDETKESLLRGSKRINLNYYPICPNPELTVGVGRHSDVSTLTILLQ

[0136] DDIGGLYVRGNNDSWVHVPPVSGSLVINVGDALQIMSNGRYKSIEHRVVASGSKNRISVPIFVNPRPCDMIGPFLEVLA RGEKALYKQVLYSDYVKHFFRKAHDGKNTIEFAKI

[0137] SEQ ID NO: 14 Populus trichocarpa: Potri.001G006901 (67.7% identity with SEQ ID NO: 9)

[0138] MAPTLAVSFNDSSDITDFVLNKGNGVKGLSEMGLESLPKQYIQPLEERMCGTKIMSHESIPIIDMSKWDDPKVAEAICE

[0139] AAEKWGFFQIINHGVPIEVLENVKEATHQFFRLPAEEKRKYLKEFSPSNNVRFGTSFSPEAEKALEWKDYLSLFYVSEDE

[0140] ASALWPAVCKDQVLEYMKRSETVIRKLLDVLM KNLNVTEIDETKESLLMGSKRTNLNYYPICPNPELTVGVGRHSDVST

[0141] LTFLLQDDIGGLYVRGNNDSWIHVPPVSCSIVINVGDALQIMSNGRYKSIEHRVIANGSNNRISVPIFINPMPSDKISPFP EVLAGGEKAVYKEVLYSDYVKHFFRKAHDGKKTIDLAKI

[0142] SEQ ID NO: 15 Theobroma cacao: Thecc.09G209400 (65.1% identity with SEQ ID NO: 9)

[0143] MAPTIADQLFNTDSSALTDFVINQGNGVKGLSEMGLKALPKAYIQPLEERMWASATQVIPEESIPIIDMTNWEDPKVA

[0144] KAVCDAAEKWGFFQIVNHAVPIQVLENVQDATHRFFGLPAEEKKKYSKEHSASSNVRFGTSFSPKAEKALEWKDFLSLF

[0145] FVSEEEACAFWPPVCREQVLEYM RSSEVVIKQLFQILMNGLNVKEIDETTKSLLMGSVRTNLNYYPICPNPELTVGVGR

[0146] HSDVSSLTILLQDEIGGLYVKGNQGGNWIHVPPIKGSLVINVGDALQILSNGRYRSVEHRVIANGSKNRISVPIFVNPRPG DIIGPLPQVLENGQKPIYKQVLYSDYVRHFFHKAHDGKKTVEFAEI

[0147] SEQ ID NO: 16 Eucalyptus grandis: Eucgr.K03272 (36.7% identity with SEQ ID NO: 9)

[0148] MKQTLPSHGQLITPCDLDFDRVKELQEFDETKAGVKGLVDSGATKVPRIFIHPHENSQSPPSDANGTSLQIPIIDLRGYR

[0149] DSRRKDVINGIRKASEAWGFFQIINHGIPLDVMDNTLEGVKKFHEQRVEVKKEVYSRDGAKRVRYFCSGDLFALKAAT

[0150] WKDTWFEFQDGDLEPEAVPPICRDAVFEYEKHIAELKTSLAELLSEALGLSSDYLSGTECMKSETVIGHYYPACPEPELTL GTVHHSDLSFLTLLLQDHHGGLQVLHESHWLDVVPVKGALLANIGDFMQLVTNGKFKSVEHRVLSGQVGPRISVACFL APSKTGKNRPLGPIKELLSENNPPIYKETSLSEYVACFRSHGLRATSTLPHFRMSDSK

[0151] F6'H2-sequences

[0152] SEQ ID NO: 17 Arabidopsis thaliana

[0153] MNQTLAAQFLTRDQVTNFVVHEGNGVKGLSETGIKVLPDQYIQPFEERLINFHVKEDSDESIPVIDISNLDEKSVSKAVC

[0154] DAAEEWGFFQVINHGVSMEVLENMKTATHRFFGLPVEEKRKFSREKSLSTNVRFGTSFSPHAEKALEWKDYLSLFFVSE

[0155] AEASQLWPDSCRSETLEYMNETKPLVKKLLRFLGENLNVKELDKTKESFFMGSTRINLNYYPICPNPELTVGVGRHSDVS SLTILLQDEIGGLHVRSLTTGRWVHVPPISGSLVINIGDAMQIMSNGRYKSVEHRVLANGSYNRISVPIFVSPKPESVIGPL LEVIENGEKPVYKDILYTDYVKHFFRKAHDGKKTIDFANI SEQ ID NO: 18 Brassica carinata: BcaC01g03987 (79.2% identity with SEQ ID NO: 17)

[0156] MAPTLSTVQFADPAEVTEFVVNKGNGVKGLSETGIKALPDQYIQPFEERLINKFVNETDEAIPVIDMSNPEEDKVAEAV CDAAERWGFFQVINHGVPLEVLDNVKAATHRFFNLPVEEKSRFTRENSLSTNVRFGTSFSPRAEKALEWKDYLSLFFVS ETEAEQYWPDACKNEALEYMNKSKTMVRKLLEYLGKNLNVKELDKTKESLFMGSIRINLNYYPICPNPDLTVGVGRHS DVSSLTILLQDQIGGLHVRSLSSGNWVHVPPVPGSFVINIGDAMQILSNGRYKSVEHRVLANGSNNRISVPIFVNPKPES

[0157] VIGPLPEVVANGEEPVYRDVVYSDYVRYFFKKAHDGKKTIDFAKI

[0158] SEQ ID NO: 19 Arabidopsis thaliana: AT3G13610 (77.4% identity with SEQ ID NO: 17)

[0159] MAPTLLTTQFSNPAEVTDFWYKGNGVKGLSETGIKALPEQYIQPLEERLINKFVNETDEAIPVIDMSNPDEDRVAEAVC

[0160] DAAEKWGFFQVINHGVPLEVLDDVKAATHKFFNLPVEEKRKFTKENSLSTTVRFGTSFSPLAEQALEWKDYLSLFFVSE AEAEQFWPDICRNETLEYINKSKKMVRRLLEYLGKNLNVKELDETKESLFMGSIRVNLNYYPICPNPDLTVGVGRHSDV SSLTILLQDQIGGLHVRSLASGNWVHVPPVAGSFVINIGDAMQIMSNGLYKSVEHRVLANGYNNRISVPIFVNPKPESV IGPLPEVIANGEEPIYRDVLYSDYVKYFFRKAHDGKKTVDYAKI

[0161] SEQ ID NO: 20 Prunus persica: Prupe.6G006000 (69.2% identity with SEQ ID NO: 17)

[0162] MVPTMTSPSGITNFVINNGNGVKGLSEMGLKSLPKQYIQPLEERISTASFGIKNSEETVPIIDMSNWDDPKVAEAICSAA EKWGFFQLVNHGVPVEVLENVKEATHRFFELPAEEKSKNSKERSCSNNVRFGTSFSPQAEKALEWKDYLSLFYVSEDEA SALWPPACKDEVLEYMKKSEILIKRLLEMLMKRLNVKEIDQEKEGLLMGSM RINLNYYPICPNPELTVGVGRHSDVSTLT VLLQDQIGGLYVRGGTDKKTWIHVPPVKGSLVINIGDALQIMSNGRYKSIEHRVAANGSKNRISVPIFVNPRPSDLISPLP

[0163] EVLASGEKAVYKQVLYSDYVKHFFRKAHDGKSTIEFAKI

[0164] SEQ ID NO: 21 Quercus lobata: QL01p008285 (67.3% identity with SEQ ID NO: 17)

[0165] MAPTIATPISESSNITDFVVTNGNGVKGISEMGLKTLPKQYIQPVEERITVSNILPQESIPIIDMSNWDEQKVSESICDAA EKWGFFQIINHGVPIEVLENVKVATHRFFNLPAEEKRKFSKENSPSNSVRFGTSFSPEAEKALEWKDYLSLFYVSEDEVSA LWPSACKDQVLEYMRGSELVIQRLLEALMKRINVKEIDETKESLLRGSKRINLNYYPICPNPELTVGVGRHSDVSTLTILLQ DDIGGLYVRGNNDSWVHVPPVSGSLVINVGDALQIMSNGRYKSIEHRVVASGSKNRISVPIFVNPRPCDMIGPFLEVLA

[0166] RGEKALYKQVLYSDYVKHFFRKAHDGKNTIEFAKI

[0167] SEQ ID NO: 22 Populus trichocarpa: Potri.001G006901 (66.6% identity with SEQ ID NO: 17)

[0168] MAPTLAVSFNDSSDITDFVLNKGNGVKGLSEMGLESLPKQYIQPLEERMCGTKIMSHESIPIIDMSKWDDPKVAEAICE

[0169] AAEKWGFFQIINHGVPIEVLENVKEATHQFFRLPAEEKRKYLKEFSPSNNVRFGTSFSPEAEKALEWKDYLSLFYVSEDE ASALWPAVCKDQVLEYMKRSETVIRKLLDVLM KNLNVTEIDETKESLLMGSKRTNLNYYPICPNPELTVGVGRHSDVST LTFLLQDDIGGLYVRGNNDSWIHVPPVSCSIVINVGDALQIMSNGRYKSIEHRVIANGSNNRISVPIFINPMPSDKISPFP EVLAGGEKAVYKEVLYSDYVKHFFRKAHDGKKTIDLAKI

[0170] SEQ ID NO: 23 Eucalyptus grandis: Eucgr.K00040 (37.5% identity with SEQ ID NO: 17)

[0171] MEAKVISGGTRFTSLPRSYVRPESERPRLFEVSAFEHVPIIDLGCNDRSRVVRQVGDACRVYGFFQVINHGVSTEAVER MQEVAAEFFRLPEEEKMKLYSEDPTKTM RLSTSFNVKKEKVHNWRDYLRLHCHPLEKYM EEWPANPPTFKEFVSNYC REVRRLGYRLEELISESLGLEKDAVRNILGEQGQHMAVNFYPPCPEPELTYGLPGHTDPNALTILLQDLHVAGLQVLKDG KWVAIDPHPNAFVINIGDQLQALSNGRYKSVWHRAIVNADKPRMSIASFLCPSDDALISSPVSLLDNGCGPTYRDFTYA

[0172] EYYKKFWSRNLDQEHCLELFKNQA

[0173] GSG linker sequence

[0174] SEQ ID NO: 24 ggatctgga

[0175] T2A sequence

[0176] SEQ ID NO: 25 gagggcagaggaagtctgctaacatgcggtgacgtcgaggagaatcctggccca Materials and methods

[0177] Plant material and vector construction

[0178] Populus tremula x Populus alba INRA-clone 717-1B4 was used as a control and for plant transformation. For poplar transformation, three different vectors were designed. Vector 1 comprises the coding sequence of Arabidopsis COSY (AT1G28680) (SEQ ID NO: 2), lacking a stop codon, fused to a glycine- serine-glycine (GSG) spacer (SEQ ID NO: 24) and a T2A linker sequence (SEQ ID NO: 25) (Halpin et al., 1999; Wang et al., 2015). In turn, the T2A linker is fused to the coding sequence of Arabidopsis F6'H1 (AT3G13610) (SEQ ID NO: 9). Vector 2 contains the two coding sequences in the opposite order, separated with a GSG spacer and T2A linker sequence. Vector 3 only contains the coding sequence of F6'H1. The resulting constructs, flanked by AttLl and AttL2 sites (Construct 1: AttLl-COSY:GSG:T2A:F6'Hl-AttL2, Construct 2: AttLl-F6'Hl:GSG:T2A:COSY-AttL2 and Construct 3: AttLl-F6'Hl-AttL2) were synthesized and cloned into the pUC57-Kan vector by GenScript. The CELLULOSE SYNTHASE 8-B promoter (pCesA8-B) (SEQ ID NO: 1) from P. trichocarpa was previously cloned in a pUC57-Kan vector (De Meester et al., 2022b). The expression vectors were created by an LR multisite II reaction with donor vectors pUC57-pCesA8-B and pUC57-COSY:GSG:TA:F6'Hl or pUC57-F6'Hl:GSG:T2A:COSY or pUC57-F6'Hl vectors into the destination vector pK7m24GW. The expression vectors pCesA8B:COSY_F6'Hl (SCOP1), pCesA8B:F6'Hl_COSY (SCOP2) and pCesA8B:F6'Hl (SCOP3) were introduced into Agrobacterium tumefaciens strain C58C1 PMP90, via electroporation. Positive colonies were selected via PCR using the following gene-specific primers: pCesA8B_fw (5'-ATGATTGATGATTATGGGAACCATGCC-3') (SEQ ID NO: 26), COSY_rv (5'-CTGTCCCAAACCGGTTCGATC-3') (SEQ ID NO: 27) and F6'Hl_rv (5'- GTTGATCACAAAAGATCCAGCAACC-3') (SEQ ID NO: 28). Agrobacterium-mediated transformation of Populus tremula x P. alba 717-1B4 was performed according to Brasileiro et al. (1992).

[0179] Growth conditions

[0180] For the first batch, 30 independent transgenic lines (one plant for each line; 16 SCOP1 lines, 13 SCOP2 lines and 1 SCOP3 line) and WT controls (seven plants) were transferred from in vitro culture to soil in pots of 5.5 cm diameter, placed in a tray filled with water and covered with a cage liner (Tecniplast APET disposable cage liner for cage body 1291H) for acclimatization. After one week, one side of the cage liner was lifted and kept accordingly for two days after which the other side was also lifted to gradually reduce humidity. Subsequently, the cage liner was completely removed, allowing the plants to continue their growth for one week in growth chambers. Afterwards, the plants were transferred to 3 L pots and placed within the greenhouse.

[0181] After four weeks of growth in the greenhouse (16 h light / 8 h dark photoperiod at +- 21°C), the plants were transferred to 10 L pots filled with N.V. Van Israel soil. For the second batch, 100 plants (five plants per independent line; 9 SCOP1 lines (4 selected from batch 1 and 5 new lines), 9 SCOP2 lines (5 selected from batch 1 and 4 new lines) and 1 SCOP3 line) and WT controls (ten plants) were transferred from in vitro culture to soil.

[0182] Harvesting of plant material

[0183] For both batches, the wild types reached heights of approximately 200 cm after 23 weeks of growth in soil. At this point, all poplars were harvested by cutting the stem 12 cm above the soil level leaving 2-3 axillary buds to allow development of new shoots. The bottom 10 cm of the harvested stem were debarked, the wood cut in pieces of about 1 cm, frozen and stored in -70°C for phenolic profiling. The leftover stem piece was weighed, debarked, air-dried, weighed, and ground in a ball mill for cell wall analysis and saccharification.

[0184] Phenolic profiling

[0185] Soluble phenolic compounds were extracted from approximately 15 mg of stem material with 1 mL methanol at room temperature for 15 min under 750 rpm shaking. After centrifugation at room temperature and maximum speed 800 pL of supernatant was transferred in a new Eppendorf and dried at reduced pressure in a SpeedVac and subsequently solubilized in 100 pL of cyclohexane and 100 pL Milli-Q. water. Samples were subjected to Ultra Performance Liquid Chromatography High Resolution Mass Spectrometry (UPLC-HRMS) at the VIB Metabolomics Core Ghent (VIB-MCG). 10 ul was injected on a Waters Acquity UHPLC (Waters) device connected to a Vion HDMS Q-TOF mass spectrometer (Waters). Chromatographic separation was carried out on an ACQUITY UPLC BEH C18 (150 x 2.1 mm; 1.7 pM) column (Waters), column temperature was maintained at 40°C. A gradient of two buffers was used for separation: buffer A (water + 0.1% formic acid, pH 3) and buffer B (acetonitrile + 0.1% formic acid, pH 3), as follows: isocratic flow at 99% A for 0.5 min, decreased to 50% A in 30 min, decreased to 30% from 30 to 35 minutes, and decreased to 0% from 35 to 37 minutes. The flow rate was set to 0.35 mL / min. Electrospray Ionization (ESI) was applied, LockSpray ion source was operated in negative ionization mode under the following specific conditions: capillary voltage, 2.5 kV; reference capillary voltage, 2.5 kV; source temperature, 120°C; desolvation gas temperature, 550°C; desolvation gas flow, 800 L / h; and cone gas flow, 50 L / h. The collision energy for full MS scan was set at 6 eV for low energy settings, for high energy settings (HDMSe) it was ramped from 20 to 70 eV. For DDA-MSMS the low mass ramp was ramped between 15-35 eV, and the high mass ramp was ramped between 35-70 eV. Mass range was set from 50 to 1500 Da in full MS with a scan time of 0.1 s and from 100 to 1500 Da in DDA-MSMS with a scan time of 0.2 s. Nitrogen (greater than 99.5%) was employed as desolvation and cone gas. Leucine-enkephalin (100 pg / pL solubilized in watenacetonitrile 1:1 [v / v], with 0.1% formic acid) was used for the lock mass calibration. Profile data was recorded through Unifi Workstation v2.0 (Waters). Data processing was done with Progenesis QI v3.0 (Waters). Data processing was performed with Progenesis QI software version 3.0 (Waters) for chromatogram alignment and compound ion detection. The detection limit was set at maximum sensitivity. Data is normalized to all compound ions. In ESI- ionization, 37286 compound ions were detected and aligned to "PooledSample_02". From the resulting chromatograms, 15230 deisotoped peaks were integrated and aligned via Progenesis QI v2.4 (Waters Corp., Milford, MA, USA), each peak having an m / z and a retention time. The following filters were applied to analyze the data: average ion intensity within at least one group >500, P value Student's t test <0.01, and fold change >2 or <0.5 compared to WT. Statistical analyses were performed on ArcSinh-transformed ion intensities. For structural elucidation, MS / MS was used.

[0186] Cell wall preparation

[0187] The preparation of CWR involved a series of sequential extraction steps. In summary, ground wood powder was sequentially washed for 30 min each with Milli-Q water at 98°C, ethanol at 76°C, chloroform at 59°C and acetone at 54°C. The remaining CWR was dried under vacuum and was determined gravimetrically (expressed as mass percentage of dry weight).

[0188] Lignin quantification

[0189] The lignin content was determined using the gravimetry-based Klason assay. Klason lignin was determined on 50 mg of ground wood powder per sample and performed according to National Renewable Energy Laboratory (Sluiter et al., 2012). Briefly, 0.5 mL of 72% sulphuric acid was added to 50 mg of CWR in glass vials and stirred with a glass rod while incubating at 30°C for 1 hour. Next, 14 mL water was added to reach a final concentration of 4% sulphuric acid. These samples were subsequently transferred to 50 mL bottles and are autoclaved for lh at 121°C. After autoclaving, these samples were stored at 4°C for 16 hours. Following that, 1 mL of supernatant was collected to determine acid-soluble lignin while the acid insoluble lignin was filtered and washed using pre-weighed glass microfiber filter papers (Sartorius AG) in Buchner filter system (Merck Millipore). Subsequently, the filters were dried in an oven at 105°C for 16 hours. These filter papers contain both lignin and ash content and were weighed using an analytical balance (XPE105; Mettler-Toledo). Next, the filter papers were transferred to a muffle furnace following a specific temperature program (12 min at 105°C, 10°C / min to 205°C, 30 min at 250°C, 20°C / min to 575°C, 180 min at 575°C, cool down at room temperature in a desiccator for 1 hour) and weighed to calculate the ash-corrected lignin content. The acid-soluble lignin was determined using a spectrophotometer (Genesys 10 S UV-VIS, Thermo-Scientific) by measuring absorbance at 205 nm. The acid-soluble lignin content was calculated with the Lambert-Beer law (Dence, 1992). Saccharification assays

[0190] The saccharification assay was performed according to (Van Acker et al., 2016). In short, 10 mg of wood powder was weighed in individual Eppendorf using an analytical balance (XPE105; Mettler-Toledo). Different biomass pretreatments were performed, alkaline pretreatment (62.5 mM NaOH) and acid pretreatment (1 M HCI). Afterwards, the biomass (pretreated as well as not pretreated) was washed by adding 1 mL of 70% EtOH which was incubated for 17 hours at 55°C. Next, the pellet was washed another three times by each time adding 1 mL 70% EtOH and washed a last time with 1 mL acetone. The pellet was afterwards dried in a SpeedVac. The actual saccharification starts by adding 1 mL of acetic acid buffer (pH 4.7) and 100 pL of 100 times diluted cellulase enzyme mixture (Cellic CTect2, Sigma) to the dried biomass at 50°C. The activity of the enzyme mixture was determined using a filter paper assay, and 0.005 and 0.007 filter paper units were added to each sample in the first and second saccharification assay, respectively. Glucose release was determined at five different time points (2h, 6h, 24h, 48h and 72h after enzyme addition) using GOD-POD where glucose was determined using a spectrophotometer by measuring absorbance at 405 nm after which the concentration was calculated using the Lambert-Beer law.

[0191] References

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[0197] Dence, C. W. (1992). The Determination of Lignin BT - Methods in Lignin Chemistry. In (ed. Lin, S. Y.) and Dence, C. W.), pp. 33-61. Berlin, Heidelberg: Springer Berlin Heidelberg.

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Claims

Claims1. A multi-cistronic chimeric expression construct comprising a plant expressible promoter operably coupled to a nucleotide sequence encoding a coumarin synthase enzyme, a nucleotide sequence encoding a 2A peptide, a nucleotide sequence encoding a feruloyl-CoA-6'-hydroxylase enzyme and a plant terminator sequence.

2. A multi-cistronic chimeric expression construct according to claim 1 wherein said plant expressible promoter is a plant promoter active in cells developing a secondary cell wall.

3. A multi-cistronic chimeric expression construct according to claims 1 or 2 wherein said plant expressible promoter is the cellulose synthase 8-B promoter.

4. A multi-cistronic chimeric expression construct according to claims 1, 2 or 3 wherein said feruloyl- CoA-6'-hydroxylase enzyme is a feruloyl-CoA-6'-hydroxylase-l enzyme or a feruloyl-CoA-6'- hydroxylase-2 enzyme.

5. A multi-cistronic chimeric expression construct according to anyone of claims 1 to 4 wherein the 2A peptide is a T2A peptide.

6. A woody plant cell, a woody plant seed or a woody plant comprising a multi-cistronic chimeric expression construct according to any one of claims 1 to 5.

7. The use of a multi-cistronic chimeric expression construct according to any one of claims 1 to 5 to increase the saccharification and pulping efficiency of woody plants.

8. A method for producing a woody plant with increased saccharification and pulping potential as compared to a corresponding wild type woody plant, whereby the method comprises transforming a multi-cistronic chimeric expression construct according to anyone of claims 1 to 5.

9. A method for the manufacture of wood pulp, said method comprising preparing a genetically modified woody plant as defined in claim 6, obtaining wood from the said woody plant and converting the said wood into wood pulp.

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