WO2002050294A1

WO2002050294A1 - A novel tissue specific plant promoter

Info

Publication number: WO2002050294A1
Application number: PCT/DK2001/000841
Authority: WO
Inventors: Knud Larsen
Original assignee: Danmarks JordbrugsForskning
Current assignee: Danmarks JordbrugsForskning
Priority date: 2000-12-19
Filing date: 2001-12-19
Publication date: 2002-06-27
Anticipated expiration: 2003-06-19
Also published as: AU2002221582A1; WO2002050294A9

Abstract

The present invention relates to the field of plant molecular biology, more particularly to regulation of gene expression in plants, and more particularly to regulation of gene expression in lignin-producing tissues in plants. The objects of the invention are achieved through the use of a novel tissue specific promoter, namely the CCR (cinnamoyl CoA:NADP oxidoreductase, EC 1.2.1.44) promoter sequence isolated from Lollium perenne, perennial ryegrass and variants of the promoter sequence. The CCR gene is a key gene in the regulation of gene expression involved in lignin biosynthesis in plants. In another aspect the invention relates to the regulation of lignin amount and/or composition in plant tissues, such as lignin-producing tissues such as the stem.

Description

A novel tissue specific plant promoter.

FIELD OF THE INVENTION

This application is a non-provisional claiming priority from Danish patent application no PA 2000 01906 filed on 19. December 2000 and PA 2001 00178 filed on 2. February 2001 , which are both hereby incoporated by reference in their entirety. All patent and non-patent references cited in these or in the present application are incorporated by reference in their entirety.

The present invention relates to the field of plant molecular biology, more particularly to regulation of gene expression in plants, and more particularly to regulation of gene expression in lignin-producing tissues in plants. In another aspect the invention relates to the regulation of lignin amount and/or composition in plant tissues, such as lignin-producing tissues such as the stem.

BACKGROUND OF THE INVENTION

Digestibility of fodder crops is determined, among other factors, by the amount of lignification which has taken place during growth of the plants and the degree of secondary modification of lignin deposited. Beside cellulose and other polysaccharides, lignins are an essential component of the call wall in tissues like the sclerenchyma and the xylem of vascular plants. Lignins play an important role in the conducting function of the xylem by reducing the permeability of the cell wall to water. They are also responsible for the rigidity of the cell wall, and, in woody tissues, they act as a bonding agent between cells, imparting to the plant a resistance towards impact, compression and bonding. Finally, they are involved in mechanisms of resistance to pathogens by impeding the penetration or the propagation of the pathogenic agent.

Lignin biosynthesis

Lignins are complex phenolic heteropolymers and, together with cellulose and hemicelluloses, constitute a major component in plant cell walls of secondary xylem and constitute the second most abundant organic compound on earth next to cellulose. Lignins provide mechanical support to plant tissues and confer hydrophobicity to vascular elements which allows the conduction of water and solutes. In addition, lignification is associated with plant responses to pathogens and mechanical stress (Moerschbacher et al., 1990; Walter, 1992).

Lignins are formed by intracellular synthesis of the monolignol precursors (p- coumaryl, coniferyl, and sinapyl alcohols), transport and secretion of the monolignols, followed by their extracellular polymerisation giving rise to the p- hydroxyphenyl, guaicyl (G) and syringyl (S) units of the lignin polymer, and coordinated with the deposition of other cell wall components (Boudet et al., 1995;

Whetten and Sederoff, 1995). The lignin biosynthetic pathway has been studied intensively, but it remains unclear which reactions control the lignin levels and composition (Whetten and Sederoff, 1995; Dixon et al., 1996; Douglas, 1996; Boudet 1998; Whetten et al., 1998). Monolignol composition varies among plant species, cell types and stage of tissue development and subcellular location (Lewis and Yamamoto, 1990). Monocototyledons use all three different monolignols and also incorporate ester- and ether-linked hydroxycinnamic acids in their lignins (H,G, and S) (Monties, 1989). In contrast, dicots primarily use coniferyl and sinapyl alcohols to produce guiacyl-syringyl lignins (G, S). Lignin biosynthesis is initiated in the phenylpropanoid pathway involving the enzymes phenylalanine ammonia-lyase

(PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate-3-hydroxylase (C3H), O- methyltransferase (OMT), ferulate-5-hydrylase (F5H) and hydroxycinnamate CoA ligase (4CL). The hydroxycinnamooyl CoA products of this pathway are the precursors of the major classes of plant phenolic compounds including lignin. Two reductive enzymes catalyze the conversion of the hydroxycinnamoyl-CoA esters into monolignols. The involved enzymes cinnamoyl-CoA reductase (CCR) and cinnamoyl alcohol dehydrogenase (CAD) are considered to be specific for ligninfication. The utilisation of inhibitors specific for these two enzymes indicates that they regulate the quantity of lignin rather than its composition.

Attempts to regulate lignin content and/or composition through regulation of enzymes located prior to CCR in the lignin biosynthesis pathway are likely to interfere with the other pathways, in which the lignin precursors are involved. This may cause undesired and fatal side effects for the plants. Importance of lignin

Lignins are not only important in the productivity and performance of field crops but are also of great importance in trees for paper making. Considerable energy and chemical input is required to loosen, dissolve and remove lignin from the cellulose fibre which is required for paper making.

In addition to these instances in which lignins present a constraint on the use of crop plants, lignins are also used for the preparation of speciality chemicals such as phenolics which can be used as precursors in chemical synthesis. Lignin confers strength on the tissues in which it is deposited. Therefore, in some aspects the presence of lignin is desirable and it may even be an advantage to increase the amount of lignin to increase the strength of the fibres. This is especially important when plant fibres are used for construction, for furniture, in plant fibre based products such as MDF plates (medium density fibreboard) and in novel composite products comprising mixtures of plant fibres and synthetic compounds. Thus lignins and their biological and chemical modification are important.

Manipulation of lignin content

Plants with a reduced amount of lignin or modified lignin composition would be more efficiently used as a forage for cattle. The yield or milk and meat would be therefore increased. Furthermore, lignin may have a negative effect on plant growth. Thus, a reduction of the lignification in crops such as wheat, oilseed rape, sugar beet or maize might presumably increase the grain yield. Trees with reduced lignin contents or altered lignin structure will lead to a reduction in the cost of the paper as less lignin will have to be removed during the pulping process. On the other hand,, novel papers may be produced due to the purity of cellulose fibre which could otherwise not be produced.

The principal applications of the present invention are improvement of the digestibility of forage crops, reduction of lignin in wood for cellulose fibre extraction, improvement of the response of crop plants to pathogen attack, and, improvement of plant fibre and thereby also timber quality. Some of these applications may require that the total amount of lignin be reduced; others may require that the amount of lignin be increased. It may also be the case that alteration of the chemical composition of the lignin polymer will confer advantages in the selected application.

Industrial processes for the extraction of cellulosic fibres from wood amount in essence to a chemical extraction procedure for removing lignin. Once lignin is removed from the wood the cellulosic fibres may be recovered and manufactured into paper or utilised in other ways, for example the cellulose may be further processed into cellulosic films or yarn for weaving or knitting into fabrics. Reduction of the lignin synthesised by the plants used for cellulose fibres, trees usually, will have a direct effect of reducing the chemical and energy demands of such extractive processes and reduce the amount of effluent material which are well-recognised as a major potential environmental pollutant which is both difficult and expensive to process. Alteration of the chemical composition of the lignin will potentially alter the solubility characteristics of the lignin in the chemical extractants used. Again this should lead directly to a reduced usage of chemicals and lower energy requirements. Finally, alteration of the lignin quality of presently unsuitable species may provide alternative sources for the papermaking industry and the cut timber industry.

Anti-sense technology

Reduction of lignification can be achieved using antisense RNA. Antisense RNA technology is an appropriate molecular biology approach to the inhibition of lignification. An antisense RNA is generated by the transcription of the non-coding DNA strand (nonsense). Thus, antisense RNA has the same sequence as the coding DNA strand and is complementary to the mRNA product of a specific gene.

As is well known, a cell manufactures protein by transcribing the DNA of the gene for that protein to produce RNA, which is then processed (e.g. by the removal of introns) into messenger RNA and finally translated by ribosomes into protein. This process may be inhibited by the presence in the cell of "antisense RNA". Therefore, as used herein, the term "antisense RNA" means an RNA sequence which is complementary to a sequence of bases in a RNA: complementary in the sense that each base (or a majority of bases) in the antisense sequence (read in the 3" to 5' sense) is capable of pairing with the corresponding base (G with C, A with U) in the mRNA sequence read in the 5' to 3' sense. It is believed that this inhibition takes place by formation of a duplex between the two complementary strands of RNA, preventing the formation of protein. How this works is uncertain: the RNA duplex may interfere with further transcription, processing, transport or translation, or lead to degradation of the mRNA, or have more than one of these effects. Such antisense RNA may be produced in the cell by transformation with an appropriate DNA construct arranged to transcribe backwards part of the coding strand (as opposed to the template strand) of the relevant gene (or of a DNA sequence showing substantial homology therewith).

The use of this technology to downregulate the expression of specific plant genes has been described, for example in EP 0 271 988 (ICI). Thus antisense RNA has been proven to be useful in achieving down-regulation of gene expression in plants.

CCR

Cinnamoyl CoA:NADP oxidoreductase (CCR, EC 1.2.1.44) catalyzes the conversion of cinnamoyl CoA esters to their corresponding cinnamaldehydes, the first specific step in the synthesis of the lignin monomers. Cinnamoyl-CoA reductase has been purified and characterized from cambial sap of spruce (Picea abies) and from soybean (Glycine max) cell cultures (Lϋderitz and Grisebach, 1981). Molecular weights and subunit composition of spruce reductase and dehydrogenase are very similar to those of the corresponding soybean enzymes. The spruce and soybean enzymes exhibit pronounced differences in substrate specificities, which reflect the different lignin composition of gymnosperms and dicotyledenous angiosperms. It can be concluded from the kinetic constants of the enzymes that under physiological conditions feruloyl-CoA is the preferred substrate for the reductase from both sources, whereas sinapoyl-CoA is a substrate only for the soybean reductase while sinapylaldehyde is a substrate only for the soybean dehydrogenase. 4-Coumaroyl- CoA is a poor substrate for the reductase from both spruce and soybean. This result is consistent with the low content of 4-coumaryl alcohol units in gymnosperm and angiosperm lignin.

A CCR has also been purified from stems of poplar (Populus euramericana) and its physicochemical properties studied (Sarni et al., 1984). The poplar reductase consists of a polypeptide of 36 kDa whose enzymatic activity is inhibited by thiol reagents and the metal chelator 1,10-phenanthroline. This poplar CCR exhibits decreasing affinity towards feruloyl-CoA, sinapoyl-CoA and p-coumaroyl-CoA.

A CCR cDNA has also been recently isolated from Eucalyptus gunnii, a woody angiosperm (Lacombe et al., 1997). Since then, two full-length CCR cDNAs have been isolated from maize (Pichon et al., 1998). CCR sequences from sugarcane (GenBank, AJ231134), Festuca (GenBank, A47099), tobacco (GenBank, A47101), and poplar (GenBank, A47097) are also available and other putative CCR genes have been identified in the soybean, tomato, potato, Arabidopsis genomes.

Promoters for manipulation of lignin content

Attempts aimed at modulating the lignin content of plants have focused on the use of antisense constructs of enzymes in the lignin biosynthesis pathway under the control of various known promoters. . Examples of combinations of promoter sequences and anti-sense constructs used for this purpose include the 35S promoter from Cauliflower mosaic virus or the GPAL3 or GPAL3 promoter with CAD from tobacco (US 6,066,780, WO 93/05159); CCR from alfalfa, maize, poplar, fescue, and eucalyptus under the control of the 35S promoter (WO 97/12982, US

6,015,943); the C4H promoter isolated from Arabidopsis in combination with the F5H coding sequence from Arabidopsis has been expressed in Arabidopsis and tobacco (WO 98/03535); later the C4H promoter and F5H gene from Arabidopsis has been inserted into a conifer, Picea glauca-engelmanii (WO 00/46382).

One common disadvantage of the prior attempts aimed at manipulating the lignin content of plants is the widespread use of constitutive promoters, which ensure expression of the construct in all living tissues at all times and in unknown amounts (in relation to the amount of mRNA produced by the native gene to be regulated). This may lead to unpredictable results and furthermore to the synthesis of mRNA and/or enzymes undesired spatially and temporally.

Furthermore, there are no available promoters specific for the lignin biosynthesis pathway in any monocots, in any Gramineae, in any Festucoideae nor in any Lolium spp., disclosed in the prior art and there are thus no known promoters for regulation of gene expression in lignin producing tissues for this important group of plants.

It is thus an object of the present invention to provide promoter sequences suitable for use in the manipulation of lignin biosynthesis. It is a further object to provide promoter sequences suitable for control of gene expression in lignin producing tissues in plants, and in particular in monocots. It is a further object to provide constructs suitable for manipulation of lignin biosynthesis and suitable for changing the phenotype of lignin-producing tissues.

It a still further object of the invention to provide plants, in particular monocots such as forage grasses, having a modified phenotype, especially having a modified lignin content, such as a reduced lignin content. Furthermore, it is an object to provide methods for obtaining plants, constructs, vectors, and promoter sequences according to the invention.

SUMMARY OF THE INVENTION

In a first aspect the invention relates to a regulatory polynucleotide capable of promoting the expression of a coding polynucleotide sequence linked to its 3' end, selected from the group comprising:

A) the 1269 nucleotide sequence of SEQ ID NO 1

B) the corresponding 1269 nucleotides of the 1333 nucleotide sequence contained in plasmid pLPCCR deposited with the DSMZ under accession number 14003; C) a fragment comprising at least 100 contiguous nucleotides of the sequence of

SEQ ID NO 1 ;

D) a functional variant having at least 65% sequence identity to SEQ ID NO 1 , preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99% sequence identity;

D) a functional variant of the fragments of C) having at least 80% sequence identity to the fragment;

F) a polynucleotide having a 5' end and a 3' end defined respectively by a transcription start site of a Lolium perenne var. Borvi CCR and a first Sail restriction site located 5' to the transcription start site; G) a nucleotide sequence comprising at least 100 bases capable of hybridising to any one of A), B), C), D), E), or F) under conditions of low stringency, preferably under conditions of moderate stringency, more preferably under conditions of high stringency; H) the complementary polynucleotide sequence to any of the sequences of items A through G.

SEQ ID NO 1 has been isolated from the 5' upstream non-transcribed region of the CCR (cinnamoyl co-reductase) gene, more specifically from Lolium perenne var. Borvi, perennial ryegrass. SEQ ID NO 1 as well as the other disclosed sequences are capable of controlling the expression of a gene linked to their 3' end in a tissue specific way, more specifically in lignin producing tissues such as stem tissue. By tissue specific is meant that the promoter drives the transcription of a higher number of transcripts in one type of tissue than in another. Preferably the number of transcripts produced in one tissue type is at least 10 times higher than in another tissue type, more preferably at least 100 times higher, such as at least 1000 times higher, for example at least 10,000 times higher, such as at least 100,000 times higher, for example at least 1 ,000,000 times higher.

SEQ ID NO 4, which is the sequence lying 5' to the translation start site of Lolium perenne var. Borvi has been deposited with the Deutsche Sammling von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D38124 Braunschweig, Germany, on 22.01.2001 under accession number DSM 14003. The sequence has been inserted into a pCRII-TOPO plasmid (Invitrogen, Groningen, NL). SEQ ID NO 4 contains the promoter sequence of SEQ ID NO 1 and the 5' untranslated part of the CCR-gene from Lolium perenne var. Borvi.

Since CCR is regarded as the first committed enzyme in the lignin biosynthesis pathway, the regulatory polynucleotide sequences according to the invention are especially adapted for use in regulation of gene expression in lignin producing tissues, such as the expression of genes in the lignin biosynthesis pathway. These, novel regulatory polynucleotide sequences may be used for a wide variety of purposes, among these for regulating the expression of genes involved in lignin biosynthesis. According to a further aspect the invention relates to a polynucleotide comprising the 1269 nucleotide sequence of SEQ ID NO 1.

In another aspect the invention relates to 1269 nucleotide of the 1333 nucleotide sequence contained in plasmid pLPCCR deposited with the DSMZ under accession number DSM 14003.

According to another aspect, the invention relates to a polynucleotide having a 5' end and a 3' end defined respectively by a transcription start site of a Lolium perenne var. Borvi CCR and a first Sa/I restriction site located 5' to the transcription start site.

According to a further aspect, the invention relates to a CCR gene, which comprises in it's 5' non-coding region an expression signal sequence of at least 1.2 kb, wherein the promoter sequence comprises: a TATAAAT-sequence (TATA-box') at position 1182-1188; a transcription starting site downstream from the TATA-box; a startcodon at position 1334-1336; and at least one of the following regulatory - sequences: i) a CAAT sequence at position 958-961 ; ii) a ACCTAACT sequence at position 1081-1088 showing homology to box P in promoters of phenylpropanoid biosynthesis genes and to a MYB consensus binding site; iii) a TGGTAAAG sequence at position 938-945 comprising a prolamin binding box, which binds a P-box binding factor; iv) a GGATA-motif at position 439-443 being a core motif of MybSta binding site; v) a CCAACC motif at position 72-78 comprising a core of a MYB homologue binding site; vi) a GGTCAGAGGG sequence at position 910 to 919; vii) a GCAGAGTACTC sequence at position 845-855; viii) a CGATCCTCA sequence at position 697-705; ix) a CCAACAAG sequence at position 582-589; x) a CCAATCA sequence at position 957-963; xi) a GCCAGTTT sequence at position 1014-1021 ; xii) a GTAAAAGA sequence at position 820-828; xiii) a CAAAGAA sequence at position 789-795; xiv) a TGTAGCT sequence at position 561-567; xv) a GGAAC sequence at position 398-402.

The CCR genes according to the invention comprise a fully functional CCR gene capable of regulating the expression of the CCR coding region linked to the 3' end of the regulatory sequences. The regulatory sequences i) through xv) are expected to be involved in the spatial and timed expression of coding sequences linked to the promoter sequences.

In a further aspect the invention relates to a nucleotide construct comprising a promoter region comprising at least a regulatory polynucleotide as defined above and a coding region operably linked to said polynucleotide wherein the expression of said coding region changes the phenotype of a plant transformed with said nucleotide construct.

By linking the regulatory nucleotide sequences according to the invention to a nucleotide sequence comprising a coding region, is obtained a construct, which may be inserted into a host cell and express the gene coded for by the coding region. According to a preferred embodiment of the invention, the coding region comprises a gene involved in lignin biosynthesis. Thereby, the construct can be used to regulate lignin biosynthesis and thereby modify the lignin content of plants. The regulatory sequences according to the invention may also be linked to other coding sequences, such as reporter genes or resistance genes, the expression of which is desired in lignin producing tissues.

According to a further aspect the invention relates to a cloning vector comprising a nucleotide construct as described above, and in still further aspect to a transformation vector comprising said nucleotide construct. Thereby the constructs according to the invention may be cloned and maintained in a library, and they may be transformed into a suitable host cell.

Furthermore, the invention relates to a shuttle vector comprising said nucleotide construct capable of stable replication in both E. coli, A. tumefaciens, and Agrobacterium rhizogenes. According to a further aspect the invention relates to a host cell comprising a nucleotide construct according to the invention and to a host cell comprising a vector according to the invention. The host cell may either be used for stable replication of the constructs according to the invention, or the host cell may comprise a plant cell from which a complete fertile plant can be regenerated according to known techniques.

According to a still further aspect the invention relates to a transgenic plant comprising at least one nucleotide construct as described above.

By providing transgenic plants comprising at least one nucleotide construct according to the invention, plants may be obtained, which have an altered phenotype. The altered phenotype may comprise decreased lignin content, which improves the fodder quality of the plant through improved digestibility by livestock or the altered phenotype may comprise increased lignin obtained through increase in copy number of the inserted gene, whereby increased strength is obtained.

Also provided by the invention is propagation material from the plants according to the invention. The propagation material such as seeds may be used for multiplying and/or sowing the transgenic plants according to the invention.

The invention furthermore relates to a part of a plant according to the invention. Forage grasses may be eaten by the animals directly in the field, but the grasses may also be harvested, dried to hay or ensilaged and stored for later use as fodder.

Further examples of parts of plants according to the invention include parts of plants that are harvested in the field and used for other purposes such as use of the plant fibres for pulp or paper for fibre containing plates and the like or as an energy source.

Furthermore, the invention relates to a method for preparation of a polynucleotide sequence according to the invention comprising chemical synthesis of the sequences. The regulatory sequences according to the invention may also be manufactured according to known molecular biological techniques such as PCR or isolation from of a vector or a plant comprising the regulatory sequences. According to another aspect the invention relates to a method for preparation of a construct comprising insertion of a promoter region comprising a polynucleotide sequence according to the invention immediately before the start codon of a desired coding sequence.

By the inventive method is obtained a nucleotide construct which may be used for transformation of plant cells.

Furthermore, the invention relates to a method for preparation of an expression vector comprising a coding nucleotide sequence capable of being expressed in a plant cell, operably linked to a promoter region comprising a polynucleotide according to the invention, comprising insertion of said promoter region immediately before the start codon of said coding nucleotide sequence, obtaining a construct, operably inserting said construct into a plant expression vector, the expression vector further comprising a selectable marker.

According to a still further object the invention relates to a method for controlling the amount of lignin in a plant comprising the steps of, providing a construct comprising a non-coding regulatory polynucleotide according to the invention and a coding region comprising a sequence coding for an enzyme in the lignin biosynthesis pathway in sense or anti-sense orientation, inserting the construct into a plant cell, obtaining plants with an increased, with a decreased amount of lignin or with an altered lignin composition.

By the method according to the invention is provided a novel method for controlling the amount of lignin in a plant. The regulatory polynucleotide sequences according to the invention are derived from the promoter of CCR, which is the first committed enzyme in the lignin biosynthesis pathway. By coupling one of these promoter sequences with a gene in the lignin biosynthesis pathway it is ensured that the expression of the coding sequence is restricted to the tissues, in which lignin biosynthesis takes place. Thereby undesired side effects are avoided to a very large extent. According to a still further aspect the invention relates to a method for identifying new regulatory polynucleotide sequences comprising the use of an isolated polynucleotide or at least one fragment of a polynucleotide according to the invention as a probe for identification of homologous DNA sequences by screening of cDNA or genomic libraries.

According to this aspect of the invention, it is possible to identify in plant cells, further examples of regulatory sequences having a similar or identical expression pattern. These regulatory sequences may be used for identification of motifs and sequences, which confer on the regulatory sequences the ability to be expressed in lignin producing tissues during lignin biosynthesis.

Figures

Fig. 1 : Nucleotide sequence of ryegrass CCR cDNA (LpCCR) (SEQ ID NO 2) and deduced amino acid sequence (SEQ ID NO 3). The start codon (ATG) and the stop codon (TGA) are shown in bold letters. The boxed amino acid sequence indicates the putative catalytic site of CCRs. The putative NAD(P) binding domain found in other CCRs are underlined. The single letter code is used for the amino acid sequence.

Fig. 2: Multiple alignment of LpCCR with Cinnamoyl CoA reductase from other plant species obtained by Clustal W (Thompson et al., 1994). The numbers represent the position of the amino acids in the respective protein sequences. Identical amino acid residues in all sequences are indicated by asterisks. Semicolon indicate residues that are mostly conserved (identity in at least six out of eight amino acids). Dashes within a a sequence indicate gaps inserted to optimize alignment. The positions of the introns in LpCCR are indicated by black arrows. The Accession Nos of the sequences used in the comparison are: LpCCR, Lolium perenne, AF278698 (SEQ ID NO 3); FaCCR, Festuca arundinacea Schreb., A47099 (SEQ ID NO 5); SoCCR,

Saccharum officinarum, AJ231134 (SEQ ID NO 6); ZmCCRI, Zea mays, X98083 (SEQ ID NO 7); POCCR, Populus trichocarpa x P. deltoides, A47097 (SEQ ID NO 9); EgCCR, Eucalyptus gunnii, X79566 (SEQ ID NO 10, NtCCR, Nicotiana tabacum, A47101 (SEQ ID NO 11); ZmCCR2, Zea mays,Y15069 (SEQ ID NO 8). Alignments of sequences were performed using the Clustal W program on EBI WWW molecular biology server.

Fig. 3: The genomic structure of gLpCCR and the Southern blot pattern. (A) The genomic structure of gLpCCR. Exons are shown as boxed numbers and introns as thin lines. Restriction endonuclease sites for EcoRI are indicated by B=βamHI, C=C/al, El= EcoRI, EV=EcoRV, H=Hinά\\\, P=Pst\, S=Sa/l, X= nol. (B) Ryegrass genomic DNA Southern blot analysis. Genomic DNA from ryegrass (20 pg per lane) was digested with BamHI, C/al, EcoRI, H/ndlll, Sail, Xho\ and- separated on a 0.8% agarose gel and probed with the LpCCR cDNA. The sizes of the hybridising bands are indicated i kb.

Fig. 4: gLpCCR promoter sequence including the 5' untranslated region (SEQ ID NO 4). Nucleotide sequence of the 5'-flanking region of the ryegrass LpCCR gene. A 1.3 kb genomic DNA fragment flanking the 5'-end of the gene contains several regulatory elements including a TATA-like box and a CAAT box shown as underlined sequences. Potential specific transcription factors identified by PLACE. MYB sites, Box-P and PBF/Dof recognintion sites are indicated by underlined DNA sequences and by the name of the DNA binding protein. Short DNA sequences common for the gLpCCR promoter and the aspen cellulose synthase promoter are indicated by an asteriks. The sequence has been deposited with the DSMZ contained within plasmid pLPCCR (accesssion number DSM 14003).

Fig. 5: Expression analysis of LpCCR. Northern blot analysis and semiquantitative PCR of poly (A)+ RNA from different tissues of ryegrass. A. Northern blot analysis of

LpCCR gene expression. Poly(A)⁺ RNA (1 μg) from ryegrass root, stem, leaf and flower was separated on a denaturing agarose gel and transferred onto Zeta-Probe nylon filter. Hybridisation was performed with the entire LpCCR cDNA (see Materials and Methods in Example 1). B. Semiquantitative RT-PCR analysis of reverse transcribed RNA from ryegrass root, stem, leaf and flower. Five μl PCR reaction product was separated on a 1.6% agarose gel containing ethidiumbromide. The sizes of the CCR and GAPDH fragments were 1032 bp and 380 bp respectively.

Fig. 6: Phylogenetic tree of LpCCR and seven other plant CCRs. The tree was constructed using the using the clustal method of DNASTAR Megalign (DNASTAR Inc., Madison, WI) based on amino acid similarities of the full sequences. GenBank accession numbers are: LpCCR, Lolium perenne, AF278698; FaCCR, Festuca arundinacea Schreb., A47099; SoCCR, Saccharum officinarum, AJ231134; ZmCCRI , Zea mays, X98083; POCCR, Populus trichocarpa x P. deltoides, A47097; EgCCR, Eucalyptus gunnii, X79566, NtCCR, Nicotiana tabacum, A47101 , ZmCCR2,

Zea mays,Y15069.

Fig. 7: CCR constructs for transformation into Arabidopsis. The CCR gene is placed in sense (upper construct) and antisense (lower construct) under the control of the 35S promoter.

Fig. 8: CCR constructs for transformation into Arabidopsis. The CCR gene is placed in sense (upper construct) and antisense (lower construct) under the control of the ryegrass CCR promoter (pCCR).

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the invention, a nucleotide sequence is provided that allows initiation of transcription in lignin-producing tissue. The sequence of the invention comprises transcriptional initiation regions associated with lignin-producing tissues.

Thus, the polynucleotides of the present invention comprise a novel nucleotide sequence for a plant promoter, more particularly a tissue specific promoter for the gene CCR (cinnamoyl-CoA reductase).

Definitions

By "heterologous nucleotide sequence" is meant a sequence that is not naturally occurring with the promoter sequence. While this nucleotide sequence is heterologous to the promoter sequence, it may be homologous (native) or heterologous (foreign) to the plant host.

By "promoter" is meant a regulatory region of DNA usually comprising a

TATA box capable of directing RNA polymerase II to initiate RNA synthesis at the appropriate transcription initiation site for a particular coding sequence. A promoter can additionally comprise other recognition sequences generally positioned upstream or 5' to the TATA box, referred to as upstream promoter elements, which influence the transcription initiation rate. Thus the promoter region disclosed herein is generally further defined by comprising upstream regulatory elements such as those responsible for tissue and temporal expression of the coding sequence, enhancers and the like. In the same manner, the promoter elements which enable expression in the desired tissue such as the lignin-producing tissues can be identified, isolated, and used with other core promoters to confirm expression in lignin-producing tissues.

By a "functional variant" of a regulatory polynucleotide sequence is meant a polynucleotide sequence sharing some degree such as at least 65% sequence identity to SEQ ID NO 1 , preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99% sequence identity and capable of regulating the expression of a coding nucleotide sequence in the same tissues and during the same growth phase as the original polynucleotide sequence. The level of expression caused by the functional variant may be higher or lower than the level of expression caused by the original regulatory sequence.

By "homology" between two nucleotide sequences is meant sequences having a high sequence identity such as at least 65% sequence identity, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99% sequence identity.

Variations in the promoter sequence

The isolated promoter sequence of the present invention can be modified to provide for a range of expression levels of the heterologous nucleotide sequence.

Less than the entire promoter region can be utilised and the ability to drive expression in lignin-producing tissues retained. However, it is recognised that expression levels of mRNA can be decreased with deletions of portions of the promoter sequence. Thus, the promoter can be modified to be a weak or strong promoter. Generally, by "weak promoter" is intended a promoter that drives expression of a coding sequence at a low level. By "low level" is intended levels of about 1/10,000 transcripts to about 1/100,000 transcripts to about 1/500,000 transcripts.

Conversely, a "strong promoter" drives expression of a coding sequence at a high level, or at about 1/10 transcripts to about 1/100 transcripts to about 1/1 ,000 transcripts. Generally, at least about 20 nucleotides of an isolated promoter sequence will be used to drive expression of a nucleotide sequence.

It is recognised that to increase transcription levels enhancers can be utilised in combination with the promoter regions of the invention. Enhancers are nucleotide sequences that act to increase the expression of a promoter region.

Enhancers are known in the art and include the SV40 enhancer region, the 35S enhancer element, and the like.

The promoter of the present invention can be isolated from the 5' untranslated region flanking its respective transcription initiation site. The term "isolated" refers to material, such as a nucleic acid, which is : (1) substantially or essentially free from components which normally accompany or interact with the material as found in its naturally occurring environment or (2) if the material is in its natural environment, the material has been altered by deliberate human intervention to another composition and/or placed at a locus in a cell other than the locus native to the material. The sequence for the promoter region is disclosed in SEQ ID NO1.

Source of the promoter sequence

Further promoter sequences according to the invention may be isolated from any plant, including, but not limited to angiosperms, such as monocots such as grasses, such as from ryegrass (Lolium spp.), perennial ryegrass (Lolium perenne), fescue (Festuca spp.), meadowgrass (Poa spp), brome (Bromus spp.), bent-grass (Agrostis spp.) timothy (Phleum spp.), Miscanthus spp., corn (Zea mays), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), wheat (Triticum aestivum), cotton (Gossypium hirsutum), oats, barley. Preferably, plants include monocots such as ryegrass, fescue, meadowgrass, brome, bent-grass, timothy, cocksfoot, Miscanthus, corn, wheat, barley, rye, and sorghum.

Promoter sequences from other plants may be isolated according to well known techniques based on their sequence homology to the promoter sequence set forth herein. In these techniques, all or part of the known promoter sequence is used as a probe which selectively hybridises to other sequences present in a population of cloned genomic DNA fragments (i. e. genomic libraries) from a chosen organism. Methods are readily available in the art for the hybridisation of nucleic acid sequences.

Hybridisation

The entire promoter sequence or portions thereof can be used as a probe capable of specifically hybridising to corresponding promoter sequences. To achieve specific hybridisation under a variety of conditions, such probes include sequences that are unique and are preferably at least about 10 nucleotides in length, and most preferably at least about 20 nucleotides in length. Such probes can be used to amplify corresponding promoter sequences from a chosen organism by the well- known process of polymerase chain reaction (PCR). This technique can be used to isolate additional promoter sequences from a desired organism or as a diagnostic assay to determine the presence of the promoter sequence in an organism. Examples include hybridisation screening of plated DNA libraries (either plaques or colonies; see e. g. Innis et al. (1990) PCR Protocols, A Guide to Methods and

Applications, eds., Academic Press).

The terms "stringent conditions" or "stringent hybridisation conditions" include reference to conditions under which a probe will hybridise to its target sequence, to a detectably greater degree than other sequences (e. g., at least twofold over background). Stringent conditions are target sequence dependent and will differ depending on the structure of the polynucleotide. By controlling the stringency of the hybridisation and/or washing conditions, target sequences can be identified which are 100% complementary to a probe (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing).

Generally, probes of this type are in a range of about 1000 nucleotides in length to about 250 nucleotides in length.

An extensive guide to the hybridisation of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, Part I, Chapter 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, New York (1993); and Current Protocols in Molecular Biology, Chapter 2, Ausubel, et al., Eds., Greene Publishing and Wiley-lnterscience, New York (1995). See also Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y.).

Specificity is typically the function of post-hybridisation washes, the critical factors being the ionic strength and temperature of the final wash solution.

Generally, stringent wash temperature conditions are selected to be about 5°C to about 2°C lower than the melting point (Tm) for the specific sequence at a defined ionic strength and pH. The melting point, or denaturation, of DNA occurs over a narrow temperature range and represents the disruption of the double helix into its complementary single strands. The process is described by the temperature of the midpoint of transition, Tm, which is also called the melting temperature.

Formulas are available in the art for the determination of melting temperatures.

Preferred hybridisation conditions for the promoter sequence of the invention include hybridisation at 42°C in 50% (w/v) formamide, 6X SSC, 0.5% (w/v) SDS, 100 mg/ml salmon sperm DNA. Exemplary low stringency washing conditions include hybridization at 42°C in a solution of 2X SSC, 0.5% (w/v) SDS for 30 minutes and repeating. Exemplary moderate stringency conditions include a wash in 2X SSC, 0.5% (w/v) SDS at 50°C for 30 minutes and repeating. Exemplary high stringency conditions include a wash in 2X SSC, 0.5% (w/v) SDS, at 65°C for 30 minutes and repeating. Sequences that correspond to the promoter of the present invention may be obtained using all the above conditions. For purposes of defining the invention, the high stringency conditions are used.

Alignment of sequences

Methods of aligning sequences for comparison are well-known in the art.

Gene comparisons can be determined by conducting BLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al., (1993) J. Mol. Biol. 215: 403-410; see also http://www.ncbi.nlm.nih.gov/BLAST/) searches under default parameters for identity to sequences contained in the BLAST "GENEMBL" database. A sequence can be analysed for identity to all publicly available DNA sequences contained in the

GENEMBL database using the BLAST algorithm under the default parameters.

Identity to the sequence of the present invention would mean a polynucleotide sequence having at least 65% sequence identity, more preferably at least 70% sequence identity, more preferably at least 75% sequence identity, more preferably at least 80% identity, more preferably at least 85% sequence identity, more preferably at least 90% sequence identity, more preferably at least 95% sequence identity, more preferably at least 99% sequence identity, wherein the percent sequence identity is based on the entire promoter region.

The promoter sequences

Apart from the sequence set forth in SEQ ID NO 1 , the invention comprises a fragment comprising at least 50 contiguous nucleotides more preferably at least 100 contiguous nucleotides, such as for example approximately 200, 300, 400, 500 or contiguous nucleotides of the sequence of SEQ ID NO 1. It is highly likely that the promoter sequence of SEQ ID NO 1 can be modified and that specific and large portions of the sequence may be omitted without substantially altering its effect. It is also highly likely, that smaller regulatory motifs may be identified within this sequence. These smaller regulatory sequences may alone or in combination provide the same promoter effect as SEQ ID NO 1.

Also encompassed by the invention is a functional variant having at least 65% sequence identity to SEQ ID NO 1 , preferably at least 70%, more preferably at least

80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99%. Functional variants of SEQ ID NO 1 may not share a very high sequence identity with SEQ ID NO 1 but still have substantially the same effect. Parts of the sequence may be altered but still allow the regulatory proteins to bind to the sequence and thus initiate transcription.

The regulatory polynucleotide may also be described with reference to a sequence having a 5' end and a 3' end defined respectively by the transcription start site of a plant CCR and the Sa/I restriction site located 5' to the transcription start site. SEQ ID NO 1 has been sequenced using a restriction fragment cleaved with Sa/I.

Promoter sequences from other plant species, especially those that are closely related to Lolium perenne may be isolated and described using the same or other restriction enzymes to cleave fragments positioned in approximately the same location relative to the CCR transcription or translation start site. These sequences are also encompassed by the invention.

Furthermore, the regulatory polynucleotide sequences according to the invention may be described with reference to a nucleotide sequence comprising at least 20 bases, preferably at least 100 bases, capable of hybridising to any one of the sequences defined above under conditions of low stringency, preferably under conditions of moderate stringency, more preferably under conditions of high stringency, and to the complementary polynucleotide sequence to any of the sequences of the items above.

Sequence fragments

Sequence fragments with high percent identity to the sequence of the present invention also refer to those fragments of a particular promoter nucleotide sequence disclosed herein that operate to promote expression of an operably linked heterologous nucleotide sequence in lignin producing tissues. These fragments will comprise at least about 20 contiguous nucleotides, preferably at least about 50 contiguous nucleotides, more preferably at least about 75 contiguous nucleotides, even more preferably at least about 100 contiguous nucleotides of the particular promoter nucleotide sequence disclosed herein. The nucleotides of such fragments will usually comprise the TATA recognition sequence of the particular promoter sequence. Such fragments can be obtained by use of restriction enzymes to cleave the naturally occurring promoter nucleotide sequences disclosed herein; by synthesising a nucleotide sequence from the naturally occurring promoter DNA sequence; or can be obtained through the use of PCR technology. See particularly, Mullis et al. (1987) Methods Enzymol. 155: 335-350, and Erlich, ed. (1989) PCR

Technology (Stockton Press, New York). Again, variants of these promoter fragments, such as those resulting from site-directed mutagenesis, are encompassed by the polynucleotides according to the present invention.

A fragment of SEQ ID NO 1 may comprise at least nucleotides no 1-1269, for example at least no 1-1250, such as at least no 1-1230, for example at least no 1- 1210, such as at least 1 -1190, for example at least 1 -1170, such as from 1 -1150, for example at least 1-1130, such as at least 1-1110, for example at least 1-1090, such as at least 1-1070, for example at least 1-1050, such as at least 1-1030, for example at least 1-1010, such as at least 1-990, such as at least 1-970, for example at least

1-950, such as at least 1-930, for example at least 1-910, such as at least 1-890, for example at least 1-870, such as at least 1-850, such as at least 1-830, for example at least 1-810, such as at least 1-790, for example at least 1-770, such as at least 1- 750, for example at least 1-730, such as from 1-710, for example at least 1-690, such as at least 1-670, for example at least 1-650, such as at least 1-630, for example at least 1-610, such as at least 1-590, such as at least 1-570, for example at least 1-550, such as at least 1-530, for example at least 1-510, such as at least 1- 490, for example at least 1-470, such as at least 1-450, for example at least 1-430, such as at least 1-410, for example at least 1-390, such as at least 1-370, for example at least 1-350, such as at least 1-330, for example at least 1-310, such as at least 1-290, for example at least 1-270, such as at least 1-250, for example at least 1-230, such as at least 1-210, for example at least 1-190, such as at least 1- 170, for example at least 1-150, such as at least 1-130, for example at least 1-110, such as at least 1-90, for example at least 1-70, such as at least 1-50, for example at least 1-30, such as at least 1-15. Similarly a fragment of SEQ ID NO 1 may comprise at least nucleotides no 20-1269, such as at least no 40 to 1269, for example at least 60 to 1269, such as at least 80- 1269, for example at least 100 to 1269, such as at least 120 to 1269, for example at least 140-1269, such as at least 160-1269, for example at least 160-1269, such as at least 180-1269, such as at least 200-1269, for example at least 220-1269, such as at least 240-1269, for example at least 260-1269, such as at least 280-1269, such as at least 300-1269, for example at least 320-1269, such as at least 340- 1269, such as at least 360-1269, for example at least 380-1269, such as at least 400-1269, for example at least 420-1269, such as at least 440-1269, such as at least 460-1269, for example at least 480-1269, such as at least 500-1269, for example at least 520-1269, such as at least 540-1269, for example at least 560- 1269, such as at least 580-1269, for example at least 600-1269, such as at least 620-1269, for example at least 640-1269, such as at least 660-1269, for example at least 680-1269, such as at least 700-1269, for example at least 720-1269, such as at least 740-1269, for example at least 760-1269, such as at least 780-1269, for example at least 800-1269, such as at least 820-1269, for example at least 840- 1269, such as at least 860-1269, for example at least 880-1269, such as at least 900-1269, for example at least 920-1269, such as at least 940-1269, for example at least 960-1269, such as at least 980-1269, for example at least 1000-1269, such as at least 1020-1269, for example at least 1040-1269, such as at least 1060-1269, for example at least 1080-1269, such as at least 1100-1269, for example at least 1120- 1269, such as at least 1140-1269, for example at least 1160-1269, such as at least 1180-1269, for example at least 1200-1269, such as at least 1220-1269, for example at least 1240-1269.

Furthermore a fragment of SEQ ID NO 1 may comprise at least one fragment selected from the group consisting of nucleotides 1 to 20, 21-40, 41-60, 61-80, 81- 100, 101-120, 121-140, 141-160, 161-180, 181-200, 201-220, 221-240, 241-260, 261-280, 281-300, 301-320, 321-340, 341-360, 361-380, 381-400, 401-420, 421-

440, 441-460, 461-480, 481-500, 501-520, 521-540, 541-560, 561-580, 581-600, 601-620, 621-640, 641-660, 661-680, 681-700, 701-720, 721-740, 741-760, 761- 780, 781-800, 801-820, 821-840, 841-860, 861-880, 881-900, 901-920, 921-940, 941-960, 961-980, 981-1000, 1001-1020, 1021-1040, 1041-1060, 1061-1080, 1081- 1100, 1101-1120, 1121-1140, 1141-1160, 1161-1180, 1181-1200, 1201-1220, 1221- 1240, 1241-1269.

Motifs and binding sites

As described above, the sequence of SEQ ID NO 1 may comprise one or more smaller regulatory sequences that act as binding sites for factors such as MYB factors, Box P factors, MybSta factors, Box P binding factors and several other factors. A regulatory polynucleotide may thus comprises at least a translation start site and at least one regulatory sequence, such as at least two regulatory sequences, for example at least 3 regulatory sequences, such as at least 4 regulatory sequences, for example at least 5 regulatory sequences, such as at least 8 regulatory sequences, for example at least 10 regulatory sequences, such as at least 15 regulatory sequences, for example at least 20 regulatory sequences. Using knowledge about the exact length and location of the motifs having an affinity for the binding factors, it is possible to construct an artificial promoter having substantially the same tissue specificity as SEQ ID NO 1.

Furthermore the polynucleotide sequence may comprise at least one fragment selected from the group of fragments consisting of: i) a CAAT sequence; ii) a ACCTAACT sequence showing homology to box P in promoters of phenylpropanoid biosynthesis genes and to a MYB consensus binding site; iii) a TGGTAAAG sequence comprising a prolamin binding box, which binds a P-box binding factor; iv) a GGATA-motif being a core motif of MybSta binding site; v) a CCAACC motif comprising a core of a MYB homologue binding site; vi) a GGTCAGAGGG sequence; vii) a GCAGAGTACTC sequence; viii) a CGATCCTCA sequence; ix) a CCAACAAG sequence; x) a CCAATCA sequence; xi) a GCCAGTTT sequence; xii) a GTAAAAGA sequence; xiii) a CAAAGAA sequence; xiv) a TGTAGCT sequence; xv) a GGAAC sequence.

Sequences vi) to x) show homology to sequences in Eucalyptus gunnii CCR promoter sequence and sequences xi) to xv) show homology to sequences in an aspen xylem specific cellulose synthase promoter. Both of these promoter have a tissue specificity comparable to that of the present promoter sequences and the homologous sequences are likely to be of importance for the spatial and temporal expression of the genes. However, as will be noted from a comparison of the Eucalyptus gunii CCR promoter sequence to the promoter sequences of the present invention, the overall sequence similarity of the two sequences is very low.

Further nucleotides

The regulatory polynucleotide may be further modified by adding at least 20 bases located 5' to SEQ ID NO 1 in Lolium perenne, such as at least 40 bases, for example at least 60 bases, such as at least 80 bases, for example at least 100 bases, such as at least 150 bases, for example at least 200 bases, such as at least 250 bases, for example at least 350 bases, such as at least 500 bases, for example at least 750 bases, such as at least 1000 bases, for example at least 1250 bases, such as at least 1500 bases. Thereby, further regulatory sequences may be added to the promoter sequence of SEQ ID NO 1.

These additional bases may also be described with reference to the Sa/I restriction site located 5' to the translation start codon of the CCR gene in Lolium perenne. The regulatory polynucleotide may comprise additional 20 bases located 5' upstream to the Sa/I restriction site, such as at least 40 bases, for example at least 60 bases, such as at least 80 bases, for example at least 100 bases, such as at least 150 bases, for example at least 200 bases, such as at least 250 bases, for example at least 350 bases, such as at least 500 bases, for example at least 750 bases, such as at least 1000 bases, for example at least 1250 bases, such as at least 1500 bases.

Expression pattern Preferably the regulatory polynucleotide sequence is capable of promoting the expression of a coding polynucleotide sequence linked to its 3' end. More preferably, this expression is a tissue specific expression. More preferably the expression is confined to lignin producing tissue, such as stem tissue, vein tissue, root tissue, xylem tissue, wound tissue, phloem tissue or seed coat tissue.

A CCR gene

The promoter according to the invention may be described as being comprised in a CCR gene, which comprises in it's 5' non-coding region an expression signal sequence of at least 1.2 kb, wherein the promoter sequence comprises: a

TATAAAT-sequence (TATA-box') at position 1182-1188; a transcription starting site downstream from the TATA-box; a startcodon at position 1334-1336; and at least one of the following regulatory sequences: i) a CAAT sequence; ii) a ACCTAACT sequence showing homology to box P in promoters of phenylpropanoid biosynthesis genes and to a MYB consensus binding site; iii) a TGGTAAAG sequence comprising a prolamin binding box, which binds a P-box binding factor; iv) a GGATA-motif being a core motif of MybSta binding site; v) a CCAACC motif comprising a core of a MYB homologue binding site; vi) a GGTCAGAGGG sequence; vii) a GCAGAGTACTC sequence; viii) a CGATCCTCA sequence; ix) a CCAACAAG sequence; x) a CCAATCA sequence; xi) a GCCAGTTT sequence; xii) a GTAAAAGA sequence; xiii) a CAAAGAA sequence; xiv) a TGTAGCT sequence; xv) a GGAAC sequence.

These regulatory sequences i) through v) comprise known sites for binding of regulatory proteins and factors in plant cells. Preferably a CCR gene according to the invention comprises at least the regulatory sequences i), ii), iii), and iv), whose promoter effect is expected to be confined to lignin producing tissues. Sequences vi) to x) show homology to sequences in a CCR promoter from Eucalyptus gunii. Sequences xi) through xv) show homology to an aspen cellulose synthase promoter, which is active in the same tissues. These short sequences are therefore also expected to be involved in regulation of expression of a gene linked to the promoter.

The CCR gene may be isolated from an angiosperm, preferably from a monocot, more preferably from a Gramineae, more preferably from a Festuceae, more preferably from a Lolium spp, more preferably from a Lolium perenne.

Variants

Biologically active variants of the promoter sequence are also encompassed by the polynucleotides of the present invention. A regulatory "variant" is a modified form of a regulatory sequence wherein one or more bases have been modified, removed or added. For example, a routine way to remove part of a DNA sequence is to use an exonuclease in combination with DNA amplification to produce unidirectional nested deletions of double stranded DNA clones. A commercial kit for this purpose is sold under the trade name Exo-SizeTM (New England Biolabs, Beverly, Mass.). Briefly, this procedure entails incubating exonuclease III with DNA to progressively remove nucleotides in the 3' to 5' direction at 5' overhangs, blunt ends or nicks in the DNA template. However, exonuclease III is unable to remove nucleotides at 3', 4-base overhangs. Timed digests of a clone with this enzyme produces unidirectional nested deletions.

Such variants should retain promoter activity, particularly the ability to drive expression in lignin producing tissues. Biologically active variants include, for example, the native promoter sequences of the invention having one or more nucleotide substitutions, deletions or insertions. Promoter activity can be measured by Northern blot analysis, and/or by reporter activity measurements when using transcriptional fusions, and the like. Variants of SEQ ID NO 1 may be made according to known techniques by site directed mutagenesis or PCR techniques.

Nucleotide construct The nucleotide sequence for the promoter of the invention, as well as fragments and variants thereof, can be provided in nucleotide constructs or expression cassettes along with heterologous nucleotide sequences for expression in the plant of interest, more particularly in the lignin-producing tissues of the plant. Such an expression cassette is provided with a plurality of restriction sites for insertion of the nucleotide sequence to be under the transcriptional regulation of the promoter.

These expression cassettes or nucleotide constructs are useful in the genetic manipulation of any plant, when operably linked with a heterologous nucleotide sequence whose expression is to be controlled, to achieve a desired phenotypic response. By "operably linked" is meant that the transcription or translation of the heterologous nucleotide sequence is under the influence of the promoter sequence.

The nucleotide construct according to the invention comprise a promoter region comprising at least a polynucleotide according to the invention and coding region operably linked to said polynucleotide wherein the expression of said coding region changes the phenotype of a plant transformed with said nucleotide construct.

Preferably the construct comprises a coding region with a gene in the lignin biosynthesis pathway, such as a gene selected from the group comprising: PAL,

C4H, C3H, OMT, F5H, 4CL, CCR, CAD, pCCoASH, CCoAOMT. All these enzymes are involved in lignin biosynthesis and several of them have been used in manipulation of lignin content of plants.

More preferably, the construct comprises a CCR gene and/or a CAD gene. These genes code for enzymes that are regarded as being the first two committed enzymes in the lignin biosynthesis pathway. It is expected that manipulation of these genes lead to changes in overall amount of lignin in the plant rather than changes in the composition of lignin. Such changes may comprise an increase in the amount of lignin obtained though an increase in the copy number of CCR and/or CAD.

The source of the CCR and/or CAD gene is preferably the plant, into which the construct is to be inserted. The CCR gene may thus comprise a CCR gene from a plant, such as from an angiosperm, such as from a monocot, such as from a grass, such as from a Lolium spp, such as from Lolium perenne. Forage grasses constitute an important source of fodder for livestock, especially for ruminants such as cows, sheep, buffalo in the temperate regions of the world. However, it is also within the scope of this invention to provide constructs comprising the promoter regions according to the invention with any CCR and CAD gene from any type of plants including forest trees or plant fibre crops.

According to one embodiment of the invention the construct may comprise a CCR gene comprising SEQ ID NO 2, or a CCR gene comprising a sequence coding for SEQ ID NO 3. These constructs are especially adapted for transformation into forage grasses such as Lolium spp, Festuca spp, Bromus spp and more particularly into Lolium perenne. The CCR gene may also comprise a gene coding for one of the amino acid sequences in Figure 2, i.e. SEQ ID NOs 5-10.

Other genes of interest

Other categories of genes of interest for the purposes of the present invention include for example genes encoding important traits for agronomics, insect resistance, disease resistance, herbicide resistance.

Still other categories of transgenes include genes for inducing expression of exogenous products such as enzymes, cofactors, and hormones from plants and other eukaryotes as well as prokaryotic organisms. It is recognised that any gene of interest, including native coding sequences, can be operably linked to the promoter of the invention and expressed in lignin producing tissues.

Insect resistance genes may encode resistance to pests that have great yield drag such as rootworm, cutworm, European Corn Borer, and the like. Such genes include, for example, Bacillus thuringiensis endotoxin genes, U. S. Pat. Nos. 5,366,892; 5,747,450; 5,737,514; 5,723,756; 5,593,881 ; Geiser et al. (1986) Gene 48: 109 ; lectins, Van Damme et al. (1994) Plant Mol. Biol. 24: 825; and the like.

Genes encoding disease resistance traits include detoxification genes, such as against fumonosin (PCT/US95/10284); virulence (avr) and disease resistance (R) genes Jones et al. (1994) Science 266: 789; Martin et al. (1993) Science 262: 1432; Mindrinos et al. (1994) Cell 78: 1089; and the like. Anti-sense constructs

The nucleotide sequence operably linked to the promoter disclosed herein can be an antisense sequence for a targeted gene. By "antisense DNA nucleotide sequence" is meant a sequence that is in inverse orientation to the 5'-to-3" normal orientation of that nucleotide sequence. When delivered into a plant cell, expression of the antisense DNA sequence prevents normal expression of the DNA nucleotide sequence for the targeted gene. The antisense nucleotide sequence encodes an RNA transcript that is complementary to and capable of hybridising with the endogenous messenger RNA (mRNA) produced by transcription of the DNA nucleotide sequence for the targeted gene. An anti-sense sequence having this effect is in the following termed an "anti-sense equivalent" of the targeted gene. In this case, production of the native protein encoded by the targeted gene is reduced or inhibited to achieve a desired phenotypic response. Thus the promoter sequence disclosed herein can be operably linked to antisense DNA sequences to reduce or inhibit expression of a native protein in the lignin-producing tissues.

The construct according to the invention may thus comprise at least part of an anti- sense equivalent of a gene in the lignin biosynthesis pathway. Preferably the gene in the lignin biosynthesis pathway comprise a gene selected from the group consisting of PAL, C4H, C3H, OMT, F5H, 4CL, CCR, CAD, pCCoASH, CCoAOMT More preferably the gene in the lignin biosynthesis pathway is the CCR gene and/or the CAD gene.

The effect of insertion of these constructs into a plant is a down regulation of the activity of the gene in question. Preferably the antisense equivalent is capable of reducing the amount of functional gene product by at least 5 %, such as at least 10 %, for example at least 15%, such as at least 20%, such as at least 25%, for example at least 30%, such as at least 40%, such as at least 50%, for example at least 60%, such as at least 70%, for example at least 80%, such as at least 90%, for example at least 95%, such as at least 99%, for example substantially 100%. Lignin is an essential component of the plant cell walls and is responsible for the strength of plant cell walls. It is therefore expected that very high reductions in gene activities for genes like CCR and/or CAD will lead to drastic and potentially fatal reductions in the lignin amount in the plants. For the farmer and for the industry making use of plant fibres, even a small reduction in the amount of lignin in the plants is advantageous in terms of increased fodder value and/or reduced amount of energy and/or chemicals used for fibre extraction and/or bleaching.

According to one embodiment of the invention, the construct comprises at least part of the anti-sense equivalent of SEQ ID NO 2. Thereby, a reduction in the amount of CCR enzyme in the cells is obtainable. The construct may also comprise at least part of an anti-sense equivalent of a nucleotide sequence coding for SEQ ID NO 3, SEQ ID NO 5, 6, 7, 8, 9, 10, or 11.

Terminator

The expression cassette (herein also termed nucleotide construct) will also include at the 3' terminus of the heterologous nucleotide sequence of interest, a transcriptional and translational termination region functional in plants. The termination region can be native with the promoter nucleotide sequence of the present invention, can be native with the DNA sequence of interest, or can be derived from another source. Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also, Guerineau et al. (1991) Mol. Gen. Genet. 262: 141- 144 ; Proudfoot (1991) Cell 64: 671-674; Sanfacon et al. (1991) Genes Dev. 5: 141- 149; Mogen et al Plant Physiology 84: 965-968. The cassette can also contain sequences that enhance translation and/or mRNA stability such as introns.

Targeted expression

In those instances where it is desirable to have the expressed product of the heterologous nucleotide sequence directed to a particular organelle, particularly the plastid, amyloplast, or to the endoplasmic reticulum, or secreted at the cell's surface or extracellularly, the expression cassette can further comprise a coding sequence for a transit peptide. Such transit peptides are well known in the art and include, but are not limited to, the transit peptide for the acyl carrier protein, the small subunit of RUBISCO, plant 5-enolpyruvl-3-phosphikimate synthase (EPSP synthase), and the like. In preparing the expression cassette, the various DNA fragments can be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers can be employed to join the DNA fragments or other manipulations can be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction digests, annealing, and resubstitutions, such as transitions and transversions, can be involved.

Vectors

As noted herein, the present invention provides vectors capable of expressing genes of interest under the control of the promoter. In general, the vectors should be functional in plant cells. At times, it may be preferable to have vectors that are functional in E. coli (e. g., production of protein for raising antibodies, DNA sequence analysis, construction of inserts, obtaining quantities of nucleic acids). Vectors and procedures for cloning and expression in E. coli are discussed in Sambrook et al. (supra).

The transformation vector comprising the promoter sequence of the present invention operably linked to a heterologous nucleotide sequence in an expression cassette, can also contain at least one additional nucleotide sequence for a gene to be cotransformed into the organism. Alternatively, the additional sequence (s) can be provided on another transformation vector. The additional sequence(s) may comprise a marker gene, a reporter gene, or a further gene involved in lignin biosynthesis.

Vectors that are functional in plants can be binary plasmids derived from Agrobacterium. Such vectors are capable of transforming plant cells. These vectors contain left and right border sequences that are required for integration into the host (plant) chromosome. At minimum, between these border sequences is the gene to be expressed under control of the promoter. In preferred embodiments, a selectable marker and a reporter gene are also included. For ease of obtaining sufficient quantities of vector, a bacterial origin that allows replication in E. coli is preferred. Reporter and marker genes

Reporter genes can be included in the transformation vectors. Examples of suitable reporter genes known in the art can be found in, for example, Jefferson et al. (1991) in Plant Molecular Biology Manual, ed. Gelvin et al. (Kluwer Academic Publishers), pp. 1-33; DeWet et al. (1987) Mol. Cell. Biol. 7: 725-737; Goff et al. (1990) EMBO J. 9: 2517-2522; Kain et al. (1995) BioTechniques 19: 650-655; and Chiu et al. (1996) Current Biology 6: 325-330.

Selectable marker genes for selection of transformed cells or tissues can be included in the transformation vectors. These can include genes that confer antibiotic resistance or resistance to herbicides. Examples of suitable selectable marker genes include, but are not limited to, genes encoding resistance to chloramphenicol, Herrera Estrella et al. (1983) EMBO J. 2 : 987-992; methotrexate,

Herrera Estrella et al. (1983) Nature 303: 209-213; Meijer et al. (1991) Plant Mol. Biol. 16: 807-820; hygromycin, Waldron et al. (1985) Plant Mol. Biol. 5 : 103-108; Zhijian et al. (1995) Plant Science 108: 219-227; streptomycin, Jones et al. (1987) Mol. Gen. Genet. 210: 86-91 ; spectinomycin, Bretagne-Sagnard et al. (1996) Transgenic Res. 5: 131-137; bleomycin, Hille et al. (1990) Plant Mol. Biol. 7:

171176; sulfonamide, Guerineau et al. (1990) Plant Mol. Biol. 15: 127-136; bromoxynil, Stalker et al. (1988) Science 242: 419-423; glyphosate, Shaw et al. (1986) Science 233: 478-481 ; phosphinothricin, DeBlock et al. (1987) EMBOJ. 6: 2513-2518; G 418, geneticin.

Preferably, the selectable marker is selected from the group consisting of markers conferring resistance to antibiotics such as ampicillin, geneticin, hygromycin, tetracyclin, kanamycin, methotrexat G418, neomycin, or to herbicides such as glyphosate, or bialaphos.

Another group of selectable markers comprises metabolic markers. Metabolic markers typically confer the host cell with the ability to metabolise an unusual substrate that cannot be metabolised by wildtype cells, which will then not survive very long. Metabolic markers are receiving increased attention due to the growing concern related to the use of markers conferring resistance to antibiotics. The selectable marker may be selected from the group consisting of metabolic markers, such as a marker conferring the ability to metabolise compounds unnatural and/or toxic to plant cells such as 2-deoxyglucose, mannose.

Other genes that could serve utility in the recovery of transgenic events but might not be required in the final product would include, but are not limited to, examples such as GUS (β-glucoronidase), Jefferson (1987) Plant Mol. Biol. Rep. 5: 387); GFP (green florescence protein), Chalfie et al. (1994) Science 263: 802 ; luciferase, Teeri et al. (1989) EMBO J. 8: 343; and the maize genes encoding for anthocyanin production, Ludwig et al. (1990) Science 247: 449. These genes may also serve as reporter genes.

Methods for transformation

The transformation vector comprising the particular promoter sequence of the present invention, operably linked to a heterologous nucleotide sequence of interest in an expression cassette, can be used to transform any plant. In this manner, genetically modified plants, plant cells, plant tissue, seed, and the like can be obtained. Transformation protocols can vary depending on the type of plant or plant cell, i. e., monocot or dicot or gymnosperm, targeted for transformation. Suitable methods of transforming plant cells include microinjection, Crossway et al. (1986) Biotechniques 4: 320-334; electroporation, Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83 : 5602-5606; Agrobacterium-mediated transformation, see for example, Townsend et al. U. S. Patent 5,563,055; direct gene transfer, Paszkowski et al. (1984) EMBO J. 3: 2717-2722; and ballistic particle acceleration, see for example,

Sanford et al. U. S. Patent 4,945,050; Tomes et al. (1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer- Verlag, Berlin); and McCabe et al. (1988) Biotechnology 6: 923-926; Agrobacterium rhizogenes mediated transformation see for example, Tepfer M, Casse-Delbart F. Agrobacterium rhizogenes as a vector for transforming higher plants. Microbiol Sci.

1987 Jan;4(1):24-8; or Stougaard J. Agrobacterium rhizogenes as a vector for transforming higher plants. Application in Lotus corniculatus transformation. Methods Mol Biol. 1995;49:49-61 ; or Costantino P, Hooykaas PJ, den Dulk-Ras H, Schilperoort RA. Tumor formation and rhizogenicity of Agrobacterium rhizogenes carrying Ti plasmids. Gene. 1980 Oct;11(1-2):79-87. Also see Weissinger et al. (1988) Annual Rev. Genet. 22: 421-477; Sanford et al.

(1987) Particulate Science and Technology 5: 27-37 (onion); Christou et al. (1988) Plant Physiol. 87: 671-674 (soybean); McCabe et al. (1988) BioTechnology 6: 923926 (soybean); Datta et al. (1990) Biotechnology 8: 736-740 (rice); Klein et al.

(1988) Proc. Natl. Acad. Sci. USA 85 : 4305-4309 (maize); Klein et al. (1988) Biotechnology 6: 559-563 (maize); Klein et al. (1988) Plant Physiol. 91 : 440-444 (maize); Fromm et al. (1990) Biotechnology 8: 833-839; Hooydaas-Van Slogteren et al. (1984) Nature (London) 311 : 763-764; Bytebier et al. (1987) Proc. Natl. Acad. Sci,. USA 84: 5345-5349 (Liliaceae); De Wet et al. (1985) in The Experimental

Manipulation of Ovule Tissues, ed. G. P. Chapman et al. (Longman, New York), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9: 415-418; and Kaeppler et al. (1992) Theor. Appl. Genet. 84: 560-566 (whisker-mediated transformation); D. Halluin et al. (1992) Plant Cell 4: 1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports 12: 250-255 and Christou et al. (1995) Annals of Botany 75: 407-413

(rice); Osjoda et al. (1996) Nature Biotechnology 14: 745-750 (maize via Agrobacterium tumefaciens); all of which are herein incorporated by reference.

Transformation of ryegrass, which is a particularly preferred target plant according to this invention, is preferably performed by direct gene transfer - particle bombardment with an inflow gun and PEG-mediated gene uptake by protoplast. In both systems, the preferably source of target cells is embryogenic suspension cells initiated from either meristems or immature embryos. The protoplast system is highly efficient and a large number of transformants with a low copy number can be obtained. Particle bombardment is less efficient and usually gives rise to plants with multiple copies of the construct. Methods for transformation of Lolium perenne are described in further detail in Foiling 1999, "Efficient transformation of Lolium perenne L. based on PEG-mediated gene transfer to protoplasts", PhD Thesis, The

Royal Veterinary and Agricultural University of Copenhagen, Department of Agricultural Sciences, Section of Plant Breeding and Biotechnology, 49pp; and

Foiling et al 1998, Plant Science, vol 139:29-40. The contents of these publications are hereby incorporated by reference.

Methods for regeneration of plants from transformed cells are known in the art. For grasses the methods usually comprise regeneration of an embryogenic culture, embryo formation, embryo maturation, an optional desiccation step, followed by germination of the embryos and hardening of the germinated tissue culture derived plants. Methods for regeneration of plants from protoplasts of Lolium perenne are described in Foiling et al 1995, Plant Science Vol 108, 2: 229-239; Foiling and Olesen 1999, Methods Mol Biol, 111 :183-193.

Target cells suitable for transformation may preferably be selected from the group comprising pollen cells, callus cells, tissue cultures, cell cultures, protoplast cultures, plant protoplasts, plant organs, plant tissues, seeds, embryos, egg-cells, zygotes, embryogenic cultures. In the case of grasses, the preferred target cells comprise protoplast cultures and embryogenic cultures.

Whereas a main object of the invention is to provide plants having an altered expression pattern in lignin producing tissue, the invention also encompasses other host cells comprising a construct according to the invention, such as yeast cells, and bacterial cells, selected from the group comprising E. coli, Bacillus subtilis. These are mainly used for stable replication of isolated nucleotide sequences.

Transformed plants

According to the invention is also provided a transgenic plant comprising a construct according to the invention. These transgenic plants will have an altered phenotype, particularly in the lignin producing tissues, in which the coding region of the nucleotide construct is expressed. It is expected that the promoter sequences according to the invention are active in all plants across the plant kingdom.

According to a preferred embodiment of the invention, the transformed plants comprise an angiosperm, such as a monocot or a dicot.

More preferably, the invention relates to a plant being a monocot comprising a species selected from the group comprising the family Gramineae, such as the subfamily Festucoideae, such as the Festuceae tribe, such as the Lolium, Festuca Dactylis, Poa, Bromus, or Brachypodium genera. The promoter sequences according to the invention are isolated or derived from a promoter sequence in Lolium perenne, which is closely related to the other members of the Festuceae tribe, the subfamily Festucoideae and the Gramineae, the grasses. The promoter is therefore expected to be especially effective and targeted in those plants.

According to an especially preferred embodiment of the invention, the transformed plant belongs to the Lolium genus, and more particularly comprises one of the species: Lolium perenne L., Lolium multiflorum L., Lolium x boucheanum Kunth, which are commercially important ryegrasses. However, the invention also relates to all other members of the Lolium genus, particularly those listed in annex 1.

According to another preferred embodiment of the invention, the transformed plant belongs to the Festuca genus and more particularly comprises one of the species: Festuca arundinaceae L., Festuca pratensis Huds., Festuca rubra L., Festuca ovina, Festuca ovina ssp. duriuscula. The Festuca genus is closely related to the Lolium genus and the performance of a promoter base on Lolium is expected to be substantially the same in Festuca as in Lolium.

Other, related and commercially important grasses which are encompassed by the invention comprise species selected from the group comprising Dactylis glomerata L., Poa trivialis L., Poa palustris L., Poa pratensis L., Bromus catharticus, Bromus sitchensis, Miscanthus spp.

The invention is not restricted to monocots or grasses. Promoter sequences may be extremely conserved throughout the plant kingdom and there are examples in literature that promoters are active in species that are very distantly related. Other angiosperm plants, in which there is interest in manipulating the lignin content include woody species such as a Populus spp, an Eucalyptus spp, a Hevea spp, such as Hevea brasiliensis, a Quercus spp, a Betula spp, a Fagus spp, a Fraxinus spp, an Ulmus spp, a Liriodendron spp, a Liquidambar spp, a Robinia spp and fibre crops such as Cannabis sativa.

The invention furthermore extends to gymnosperms, such as conifers such as Pinus spp, for example P. taeda, P. radiata, P. silvestris, P. lambertiana, P. elliotti, P. palustris; a Picea spp such as P. abies, P. rubra, P. stichensis, P. rubra, P. mariana, P. glauca; a Larix spp, such as L. leptolepis, L. decidua, L. occidentalis, L. laricina; an Abies spp. such as A. grandis, A. fraserii, A. concolor, A. procera, A. balsamea; a Pseudotsuga spp such as P. menziesii; a Tsuga spp; a Thuja spp; a Chamacyparis spp.; a Sequoia spp. The conifers constitute a group of extremely important species for production of lumber and plant biomass for paper and pulp production. In these species there is a large interest in lowering the lignin content due to the amount of energy and hazardous chemicals used for bleaching of wood fibres.

In all the plant species listed above and in annex 1 , the coding region of the inserted construct may originate from one of the sequences according to this invention (SEQ ID NO 2 or sequences coding for SEQ ID NO 3) or it may originate from a sequence in the species in question (including the sequences set forth in SEQ ID NO 5 to 11).

After transformation and plant regeneration, the transformed plants may be further bred through crossing and selection or through generations of selfing followed by crossing and selection in order to provide new combinations of genes. It may also be advantageous to transfer the inserted constructs according to this invention from one variety to another using conventional breeding techniques, such as crossing and backcrossing, rather than through transformation of all varieties, provenances and cultivars.

In another aspect the invention relates to propagation material obtained from a transformed plant according to the invention. Propagation material such as seeds, cuttings, cell cultures, tissue cultures, zygotes, pollen, egg-cells, somatic embryos, artificial seeds, adventitious buds, axillary buds comprise a convenient means for distribution of the transformed plants. The propagation material may be made from the inventive plants using conventional propagation techniques such as seed- growing and tissue culture techniques.

The ultimate goal of the invention is to produce plant parts, comprising plant fibres, leaves, stems, wood, roots, green parts, fodder, silage, straw, hay, with an altered phenotype such as a reduced or increased level of lignin. These parts of plants are also encompassed by the invention.

The cells that have been transformed can be grown into plants in accordance with conventional ways. These plants can then be grown, and pollinated with the same transformed strain or different strains. The resulting hybrid having lignin-producing tissue expression of the desired phenotypic characteristic can then be identified. Two or more generations can be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited.

The following examples are offered by way of illustration and not by way of limitation.

Example 1: Cloning and characterization of a ryegrass (Lolium perenne) gene encoding cinnamoyl-CoA reductase (CCR)

Abbreviations:

BSA = bovine serum albumine, CCR = cinnamoyl CoA reductase, EDTA = ethylene diamine tetraacetic acid, GAPDH= glyceraldehyde-3-phosphate dehydrogenase,

PLACE = Plant c/^'s-acting regulatory DNA elements, RACE = rapid amplification of cDNA ends, SDS = sodium dodecyl sulfate.

1. Materials and Methods:

1.1 Plant material

Tissues of ryegrass (Lolium perenne var. Borvi) used in all experiments were collected from field-grown plants (Danish Insitute of Agricultural Sciences, Tjele, Denmark). Plant material used for Northern blot, Southern blot analyses and for RT- PCR was immediately frozen in liquid nitrogen after harvest and stored at -80°C.

1.2 Recombinant DNA methods

Standard DNA procedures were used (Maniatis et al., 1982; Sambrok et al., 1989).

1.3 Extraction of nucleic acids

Nucleic acids were extracted and purified from deep-frozen plant material. Isolation of genomic DNA from ryegrass leaves was carried out using a minipreparation method (Dellaporta et al. (1983). Total RNA was extracted according to the method described by Jackson and Larkins (1976). Poly(A)⁺RNA was isolated from total RNA using Dynabeads Oligo (dT)₂₅ (Dynal, Skøyen, Norway) following manufacturer's instructions. RNA concentrations were determined by UV spectrometry.

1.4 cDNA library screening, PCR cloning, and 5' and 3' RACE A solid phase cDNA micro-library was constructed using poly(A)⁺-enriched RNA isolated from stem tissue of ryegrass and Dynabeads Oligo (dT)₂₅ (Dynal, Skøyen, Norway). Briefly, 100 μg total RNA isolated from ryegrass stem tissue was primed with Dynabeads oligo-(dT)₂₅ primers, fixed onto magnetic spherical beads and reverse-transcribed using Superscript reverse transcriptase (Life Technologies). The library was reused in sequential PCR analyses of different cDNAs. CCR was amplified by PCR using oligonucleotide primers derived from conserved regions of published CCR sequences and Taq polymerase (Life Technologies).

Degenerate primers were derived from CCR sequences in the EMBL database and included oligonucleotides for the highly conserved peptide motifs in the CCR enzymes, NWYCYGK (sense, CCRs: 5'-AACTGGTATTGYTGGGGCAA-3' (Y=C/T), and QEKGH, (antisense, CCRas: 5'-ARRTGGCCYTTTTCCTG-3' (R=A/G; Y=C/T).

The following program was used for PCR amplification of LpCCR: Denaturation at

94°C for 2 min followed by 35 cycles (94°C for 30 s, 60°C for 30 s, 72°C forl min 30 s) and a final extension at 72°C for 7 min. The PCR product was cloned into pCRTOPO II vector (Invitrogen) and sequenced in both directions. The sequence data from one transformant whose plasmid appeared to contain a CCR gene fragment, was used to synthesize two exact match oligonucleotide primers, CCRsl :

5'-AACTGGTATTGTTGGGGCAAT-3' and CCRasl : 5'- AGCCTCCAGGAGAAGGGCCAT-3', used for PCR amplification of an authentic ryegrass CCR fragment.

To obtain a full-length LpCCR cDNA gene-specific sense and antisense primers derived from the isolated CCR fragment were used in 573' RACE (rapid amplification of cDNA end) experiments. In order to provide sequence of the 5' end of the library CCR clone antisense gene-specific primers were used in combination with a kit anchor primer (Roche Molecular Biochemicals). In brief, for 5'RACE a reverse transcription oligonucleotide one primer, CCRasl : 5'- AGCGTTCACCGTTGGCTGCAGCAGCGGCCC-3' was used in a reverse transcription reaction. The resultant cDNA was used as a template for PCR amplification employing the proof-reading polymerase Pfu (Stratagene), in combination with primer CCRas2: 5'-

CCACGCGGCCTGCTCCGCCACGGCCTTGCC-3' and a kit PCR anchor primer: 5'- GACCACGCGTATCGATGTCGAC-3'. In a second 5'RACE experiment a third gene- specific CCR primer CCRas4: 5'-AGGATCATCGGTGACTGGGGAGGC-3' was used for cDNA synthesis and a fourth primer CCRas4: 5'- GGCGCATATGGCATCGTAGTC-3' was used in combination with a kit anchor primer. To determine the sequence of the 3' end of the gene, 3' RACE (Roche Molecular Biochemicals) was performed following the manaufacturer's directions using a gene-specific oligonucleotide primer derived from the previously determined sequence and the kit anchor primer included in the RACE kit. The sequence of the primer used for 3'RACE (CCRs2) was: 5'-CAGAAGCTGCAGGATCTTGGC-3'. PCR products were cloned into the pCR-ll TOPO vector and sequenced on both strands. PCR products were recovered as described previously. One PCR product was shown to contain a DNA fragment that showed homology to CCR and matched the sequence of the library clone in the overlapping sequence.

Oligonucleotides were purchased from DNA Technology, Aarhus, Denmark.

1.5 Construction and screening of genomic library

Genomic DNA was isolated from leaves of Lolium perenne, partially digested with Mbo\ and size-fractionated. The selected DNA was used for construction of a genomic library in λEMBL3 (Stratagene, Palo Alto, CA). Screening of the genomic library was done by plaque hybridisation using a ³²P-labeled cDNA probe by standard procedures (Sambrook et al., 1989). Hybridisation was carried out in a solution containing 50% formamide, 5 x SSPE, 5 x Denhardt's solution, 0.1% SDS and 100 μg ml^"1 denatured salmon sperm at 65°C for 12-16 h. After hybridisation, the membranes were washed once with 2 x SSC/0.5%SDS at room temperature for 20 min., and twice with 1 x SSC/0.1 %SDS at 65°C for 10 min. each. Positive clones were purified by three to four rounds of plaque purification. Phage DNA was purified using Qiagen Lambda Midi kit (Kebo, Denmark), and analysed by restriction endonuclease digestion. Appropriate fragments were subcloned into pBluescript II SK⁺ (Stratagene) for further analysis. 1.6 DNA sequencing

PCR products, cloned cDNA and genomic DNA were sequenced by the dideoxy chain termination method using BigDye terminator cycle sequencing kit with AmpliTaq DNA polymerase FS (PE Applied Biosystems). The sequencing analysis was performed using an automated DNA sequencer (ABI PRISM™ Genetic

Analyser Model 373/377); (PE Applied Biosystems). All DNA fragments were sequenced in both directions.

1.7 Northern blot analysis and RT-PCR Aliquots of 1 μg poly(A)⁺RNA were fractionated by electrophoresis in a 1.5% agarose containing formaldehyde and Mops buffer (Sambrook et al., 1989). Subsequently the RNA was transferred onto Zeta-Probe GT membranes (Bio-Rad, Richmond, CA, USA) by vaccuum blotting and fixed by UV crosslinking according to the manufacturer's instructions. Blots were hybridised at 42°C for 18 h using a random primed ³²P-labeled NDK fragment covering only the coding region of LpCCR as probe. Radiolabeled probes were derived from gel-purified DNA fragments by random priming using pre-mixed reagents (Roche Molecular Biochemicals, Mannheim, Germany). ULTRAhyb hybridisation solution (Intermedica, Stockholm Sweden) was used in all hybridisations. Membranes were washed twice in 2 x SSC/0.5% SDS for 15 min at room temperature and twice with 0.5 x SSC/0.1 % SDS for 15 min at room temperature and finally twice with 0.1X SSC/0.1% SDS at room temperature. The membrane was stripped according to manufacturer's protocol and reprobed with a random primed ³²P-labeled 380 bp ryegrass GAPDH fragment.

Semi-quantitative RT-PCR was performed with four solid-phase ryegrass cDNA libraries constructed from root, stem, leaf and flower RNA following the manufacturer's protocol (Dynal, Skøyen, Norway). Briefly, 100 μg total RNA isolated from different ryegrass tissues were primed with Dynabeads oligo-(dT)₂₅ primers, fixed onto the magnetic spherical beads and reverse-transcribed using Superscript reverse transcriptase (Gibco, Life Technologies). Subsequently the

LpCCR cDNA was amplified using gene- specific LpCCR primers (sense primer CCRslO: 5'-ATGACTGTTGTCAACACCGTC-3', antisense primer: CCRasl 8: 5'- GAGGGATTGAACGGTGTAACGGCA-3', 1032 bp in size). For detection of ryegrass GAPDH transcript by RT-PCR, a set of gene-specific ryegrass GAPDH primers (sense: 5'-CAAGGACTGGAGAGGTGG-3', antisense: 5'- TTCACTCGTTGTCGTACC-3', 380 bp in size) was used. PCR was carried out in a reaction containing 3 μl cDNA library, 0.5 μM of the four different primers, 1.0 unit DyNAzyme II™ DNA polymerase, 0.2 mM dNTP, 1 ,5 mM MgCI₂, in a total volume of 50 μl. The following PCR program was used for PCR amplification of LpCCR and GAPDH transcripts: Denaturation at 94°C for 2 min followed by 35 cycles (94°C for

30 s, 60°C for 30 s, 72°C for 1 min 30 s) and a final extension at 72°C for 7 min. PCR products were electrophoresed on a 1.0 % agarose gel.

1.8 Southern blot analysis Genomic DNA was isolated from Lolium perenne leaves (Dellaporta et al., 1983), digested with various restriction endonucleases and fractionated on 0.8% agarose gels. Transfer of the DNA fragments to Hybond^* memebranes (Amersham- Pharmacia) was performed using a vaccum blotter and and a neutral sodiumphosphate buffer (Church and Gilbert, 1984). After transfer the DNA was fixed to the membrane by UV-crosslinking. Hybridisations were performed in a buffer containing 0.5 M sodiumphosphate pH7.2/1 % BSA/1 mM EDTA/7% SDS/50 μg ml^"1 salmon sperm DNA at 65°C using α-³²P-dATP labeled DNA fragment as a probe. This fragment, which covers the coding region of LpCCR, was labeled using a Random primed labeling kit from Roche Molecular Biochemicals. Washings were carried out at 65°C once in 2 x SSC/0.5% SDS, once in 1 x SSC/ 0.1 %SDS, and finally once in 0.1 x SSC/0.1 % SDS (30 min. each).

1.9 Bioinformatic analyses

BLASTP and BLASTN (Altschul et al., 1997) was used to identify homologues of the LpCCR protein by comparing a nucleotide sequence translated into all reading frame against protein databases (http://www.ncbi.nih.gov). Analyses of the DNA and protein sequences and structure as well as the comparative sequence analyses were carried out using a computer software DNASTAR and http://www.expasy.ch. Prediction of the secondary structure of LpCCR was performed using NNPredict (http://cmpharm.uscf.edu). Analysis of the LpCCR promoter region to search for potential regulatory c/s-acting elements was done by use of PLACE (Higo et al., 1999) accessible at http://www.dna.affrc.go.jp/htdocs/PLACE/signalscan.html.

2.1 Isolation and characterization of CCR cDNA and genomic clones from Lolium perenne A ryegrass stem cDNA library was constructed and used for PCR based cloning of a CCR gene. Degenerate oligo nucleotide primers were derived from CCR sequences from the EMBL database and used for PCR anlysis. Two highly conserved motifs in the CCR sequences, NWYCYGK and QEKGH, were selected for the synthesis of primers: CCRs: 5'-AACTGGTATTGYTGGGGCAA-3' (Y=C/T), and CCRas: 5'-

ARRTGGCCYTTTTCCTG-3' (R=A/G; Y=C/T). PCR based cloning using the solid- phase ryegrass stem library and the primers CCRs and CCRas in sense and antisense orientation respectively yielded a 500 bp DNA fragment, which was sequenced in both directions. Sense and antisense primers were designed from the determined DNA sequence of the ryegrass CCR fragment and 573' RACE experiments were carried out.

By two rounds of 5'RACE and one round of 3'RACE a near full length LpCCR gene was obtained.

The entire cDNA sequence of LpCCR consists of 1235 bp, with an open reading frame of 1035 bp, a 28 bp 5'-noncoding region and a 172 bp 3'-noncoding region (Fig. 1). The LpCCR gene encodes a protein of 344 amino acids, with a predicted molecular mass of 37.4 kDa and a pi of 6.34.

The calculated molecular mass of LpCCR 37,4 kDa agrees well with the molecular mass of other CCRs. The nucleotide sequence data reported in the present study will appear in the GenBank Database under the accession number AF278698.

A 1.6 kb LpCCR transcript was detected by Northern analysis so obviously the

LpCCR cDNA does not represent a full length clone. Additional sequence is lacking both in the 5'- and 3'-noncoding regions, e.g. no putative polyadenylation signal nor a polyA tail can be found. The nucleotide sequences found around the ATG start codon are in agreement with both the eukaryotic consensus sequence of Kozak (1984) with an A in position -3, and, as frequently observed in plants, a C in position

+5 (Lϋtcke et al., 1987).

Multiple alignment of LpCCR and other plant CCR sequences (Fig. 2) revealed several regions of high homology. An extremely conserved motif, NWYCY (pos. 165 to 169), is present in all the aligned sequences. This motif has been proposed to be directly involved in the catalytic site of CCR (Lacombe et al., 1997). The motif can be further extended to NWYCYGK with the exception of maize CCR2, which is generally different in many amino acid positions within this sequence.

Two Arabidopsis cDNA clones,AtELO1 and AtELO2, were originally identified as genes coding for CCR enzymes. However, recombinant proteins of both cDNAs expressed i E. coli failed to convert the substrates normally used by CCR enzymes and was hence judged not to represent Arabidopsis CCR. Although both AtELOI and AtELO2 exhibit high overall homology to CCR sequences there are some important differences. A comparison of the catalytic consensus sequence KNWYCY shown in Fig. 2 with the same regions in AtELOI , ENWYCL and AtELO2, ENWYSL, reveals several deviations from the consensus with two or three amino acid substitutions. In conclusion, the consensus sequence KNWYCY seems to be crucial for the enzymatic activity and hence the identity of CCR enzymes. In the N-terminal end of LpCCR between amino acid residues 20 and 40 a highly conserved motif:

VTG7_G ^A/_G ^F/_YIAS^G/_W /_MVK^I/L/_RLL^D/_E ^K/_RGY is found. This conserved sequence represents a putative NAD(P) binding domain i.e. the cofactor binding site. The predicted secondary structure of LpCCR corresponds to the βαβ-dinucleotide binding fold of NADP(H) and NAD(H) -dependent reductases and dehydrogenases.

The secondary structure of the N-terminal region of LpCCR was calculated according to Rost and Sander (1993) using NNPredict. The amino acid sequence TVCVTGAAGYIASWLVKLLLERGYTVKGTV contains a sheet-helix-sheet structure is observed which corresponds to the βαβ-dinucleotide binding fold of NAD(H) and NADP(H)-dependent dehydrogenases and reductases. An alignment of the putative

NAD(P) binding domain of Eucalyptus gunnii CCR and the bacterial enzymes cholesterol dehydrgenase and UDP-galactose 4 epimerase and the mammalian enzymes 2β-hydroxysteroid dehydrogenase demonstrated that several amino acids within this region were well conserved (Lacombe et al., 1997).

The deduced amino acid sequence of LpCCR was aligned with those of other CCRs from various plants reported in GenBank to date and their identitites were calculated (Fig. 2 and Table 1). As expected, LpCCR exhibited the highest level (about 95%) of identity to the CCR from fescue (Festuca arundicinea), like ryegrass a monocot. High levels of identity were also observed to CCRs from other monocots like sugarcane (Saccharum officinarum) and maize (CCR1). Substantial identity is also present by comparison to the CCRs from the dicots Eucalyptus gunnii, tobacco (Nicotiana tabacum) and poplar (65-74%). The lowest identity was found when comparing LpCCR to maize CCR2 indicating a different function of the latter. Similarity searches of the LpCCR cDNA were performed with the BlastnN program revealed homology to dihydroflavonol 4-reductases (DFR). The deduced amino acid sequence of LpCCR was also aligned (using ClustalW) with selected plant dihydroflavonol 4-reductases reported in GenBank and their identities were determined. Identities LpCCR to DFRs from Glycine max (GenBank, AF167556), Petunia x hybrida (GenBank, AF233639), Vitis vinifera (GenBank, X75964) and

Oryza sativa (GenBank, AB003495) ranged from 31-37 %. The high identity in this region suggests a functional identity which could be co-factor binding. The homology between LpCCR and the DFRs primarily covers the N-terminal end of the polypeptides, and in particular amino acids 20-40 in the LpCCR sequence. The homology was significantly lower outside this region.

EgCCR FaCCR LpCCR NtCCR PoCCR SoCCR ZmCCRI ZmCCR2 EgCCR 73 74 77 82 73 72 66

FaCCR 95 64 70 83 82 63 LpCCR 65 72 83 82 63 NtCCR 76 65 65 64 PoCCR 72 71 65 SoCCR 90 64 ZmCCRI 63 ZmCCR2

Table 1. Amino acid sequence identity between ryegrass CCR, and other plant CCR proteins derived from the GenBank database. The values show the percentage identities calculated using the DNASTAR Megalign program. The Accession Nos of the sequences used in the comparison are listed in legend to Fig. 2. Similarities between LpCCR and other CCR proteins are shown in bold.

2.2 Isolation of a genomic ryegrass CCR Approximately 600,000 recombinant phage were screened from a genomic ryegrass library using a 500 bp CCR PCR fragment as probe. Six CCR genomic clones of variable sizes were isolated from the Lolium perenne genomic library. Sequencing of these clones demonstrated that they all represented the same gene. The longest clone, gLpCCR, of approximately 3.2 kb were sequenced, with 1333 bp 5' nontranslated sequence, 1035 bp coding region, 2197 bp intron sequences and a 3' non-translated end. The positions of four introns (I, 81 bp, II, 102 bp, III, 919 bp and IV, 1094 bp) were deduced by comparison with the cDNA and are shown in Figure

3. The splice points conform to the GT-AG rule for donor and acceptor site (Brown, 1986). The positions of the four introns is completely identical to the localization of the four introns in the Eucalyptus gunnii genomic CCR. However, there seems to be significant differences in the length of the four introns between the two species. Introns I and IV are of comparable size while introns II and III are of totally different length. The positions of the four introns in LpCCR also perfectly matches the positions of introns in plant DFR genes.

The predicted ryegrass genomic CCR encodes 344 amino acids which are hundred percent identical with the LpCCR deduced protein sequence. At the nucleotide level, ryegrass genomic CCR and LpCCR are >95% identical for their coding region which indicates that ryegrass genomic clone corresponds to the LpCCR cDNA.

2.3 Genomic organization of ryegrass CCR To the examine copy number of CCR in the ryegrass genome, Southern analysis was conducted using total DNA from ryegrass leaves and using the coding region of LpCCR cDNA as a probe.

Genomic DNA of Lolium perenne was digested with Sa/nHI, C/al, EcoRI, Hind\\\, Sa/I and Xπol restriction enzymes. The number of restriction sites for the selected and other enzymes identified in the CCR cDNA and in the introns of the genomic CCR are shown in Fig. 3A. An EcoRI site was found in the CCR cDNA and also at the equivalent position in the third exon of gLpCCR. There were no sites of H/ndlll, EcoRI and Xho\ in the CCR cDNA, but two Pst\, two EcoRV, one C/al, one SamHI, and one HinύW sites were present in the introns of gLpCCR.

The genomic blot was hybridised with a radiolabeled probe covering the coding sequence of LpCCR. As shown in Fig. 3B one hybridising band is visible in the Sa/I and Xho\ digests whereas two hybridising bands are observed with the SamHI, C/al, EcoRI, and H/ndlll digests reflecting the presence of internal restriction sites. Two hybridising band observed with the restriction endonuclease EcoRI is explained by the internal recognition sites of the enzyme as presented in Fig. 3A. The size of the hybridising fragments agrees well with the restriction map of LpgCCR suggesting the presence of only one gene. The Southern blot analysis of total DNA i from ryegrass leaves therefore indicates that CCR is represented as a single-copy gene in the ryegrass genome (Fig.3B). The result is in accordance with the observation done with Eucalyptus gunni, CCR where only one gene was found (Lacombe et al., 1997). In contrast two cinnamoyl CoA reductase genes (CCR1 and CCR2) were observed in maize (Pichon et al., 1998).

Example 2: Analysis of the LpgCCR promoter

The nucleotide sequence of the 5'-flanking region LpgCCR gene is presented in Fig. 4. A TATA box is localized at pos. 1182 (Mitchell and Tijan, 1989). No clear consensus CAAT or CCAAT boxes are found, although four CAAT or CCAAT sequences with non-consensus flanking nucleotides are located at nucleotides 206, 328, 380, 642, 643, 957, 958 respectively (Mitchell and Tijan, 1989).

Usually the CAAT box is suggested to exist at about 70 bases upstream from the TATA box, but in the gLpCCR promoter there is much more distance (224 nucleotides) between them. Putative binding sites for transcription factors were examined using the computer-based TFSEARCH ver. 1.3 Program (by Yutaka Akiyama: http://www.rwcp.or.jp/papia/) using the transfac database (Heinemaeyer et al., 1998) and the Plant cis-acting regulatory DNA elements (PLACE) database http://www.dna.affrc.go.jp/htdocs/PLACE/ (Higo et al., 1999).

A motif, GGATA, found at position 439-443 in the ryegrass CCR promoter has been demonstrated to be a core motif of MybStl (a potato MYB homologue) binding site (Baranowskij et al., 1994). At position 72-78 a motif, CCAACC, is identified as a core of consensus maize P (MYB homologue) binding site (Grotewold et al., 1994). The maize P gene specifies red pigmentation of kernel pericarp and other floral genes. P binds to the A1 gene, but not to the Bz1 gene. Maize C1 MYB homologue activates both the A1 and the Bz1 gene (Grotewold et al., 1994). Another motif located at position 1081-1088, ACCTAACT shows striking identity to a consensus motif for sequences related to box P identified in promoters of phenylpropanoid biosynthetic genes: ^A/_CACC^T/_AA^A/_C C (Sablowski et al., 1994) and to a MYB consensus binding site ^T/_CAAC^T/_GG (Biedenknapp et al., 1988). Some of the genes encoding the enzymes of general phenylpropanoid metabolism and lignin biosynthesis, such as phenylalanine ammonia lyase (PAL), hydroxycinnamate lyase (4CL), cinnamate 4- hydroxylase, 4-coumaryl-CoA ligase, caffeic acid O-methyltransferase and cinnamyl alcohol dehydrogenase contain motifs conserved within their promoters. Some of these c/s-acting elements binds recombinant Myb protein in vitro (Sablowski et al., 1994). These motifs conform well to the motifs recognized by plant MYB transcription factors (Bugos et al., 1991; Hauffe et al., 1993; Shufflebottom et al., 1993; Sablowski et al., 1994; Ye et al., 1994; Feuillet et al., 1995; Solano et al.,

1995; Douglas et al., 1996). It is therefore likely that ryegrass MYB factors also may interact with motifs in the CCR promoter.

Furthermore, a motif of eight nucleotides.TGGTAAAG, located at pos. 938 earlier recognized as a core motif found in the maize alpha-zein promoter (Vincente- Carbajosa et al., 1997). The sequences constitutes the core of a prolamin box which binds a P-box binding factor (PBF) and binds with BPBF (barley PBF). PBF is a DNA-binding protein of the Dof class of transcription factors.

The promoter region of the LpCCR gene was compared to the 5'-flanking region of the xylem-specific cellulose synthase gene from aspen (Wu et al., 2000) and several short stretches of nucleotides with high sequence identity were found e.g. GGAAC (398-402), TGTAGCT (561-567), CAAAGAA (789-795), GTAAAAGA (820-828), TGGTAAAG (938-945), GCCAGTTT (1014-1021). Very interestingly the short sequence TGGTAAAG (938-945) was demonstrated to be the binding site of PBF/Dof suggesting that a common regulatory mechanism of LpCCR and xylem- specific cellulose synthase.

In a very recent publication Lacombe et al. (2000) present the examination of the developmental regulation of the Eucalyptus gunnii CCR EgCCR promoter by analysing the expression of EgCCR-GUS fusions in tobacco. These analyses defined a minimal promoter, between pos. -119 and -77 in the EgCCR promoter necessary and sufficient for expression in vascular tissues and in root tips. Sequence analysis of this minimal promoter revealed the presence of an AC- element with a sequence (CCCACCTACC) identical to that of the bean PAL2 promoter (Hatton et al., 1995). The ACI element present in the EgCCR promoter contains the MBSII site (G^G/_TT^A/_TGGT^A/_G), defined by Romero et al. (1998), which is bound to a subgroup of MYB factors including AmMYB308 (Tamogone et al., 1998). AmMYB acts as a repressor of phenolic acid metabolism and lignin synthesis. By comparison of the LpCCR promoter and the EgCCR promoter an imperfect sequence at pos. 910 in LpCCR, GGTCAGAGGG shows 70% identity to the ACI element, GGTAGGTGGG found in the EgCCR. It is therefore likely that this sequence also in the LpCCR promoter serves as a c/s-regulatory element. Sequence comparison of the two promoters also revealed short stretches of high sequence identity, e.g. GCAGAGTACTC (845-855), CGATCCTCA (697-705),

CCAACAAG (582-589) and CCAATCA (957-963).

Example 3: CCR gene expression in Lolium perenne

To determine the spatial expression of the CCR gene in Lolium perenne, Northern analysis was carried out with polyA⁺ RNA extracted from root, stem, leaf and flowers of ryegrass. The blot was hybridised with a CCR probe covering the coding region. Fig. 5A shows that a 1.6 kb transcript is highly expressed in stem tissue. No CCR transcript could be detected in root, leaf and flower tissue. Other enzymes involved in lignin biosynthesis like CCoAMT I family and class I COMT genes have been demonstrated to be highly expressed in most lignified tissues such as those of the basal parts of the stems or vascular vessels (Martz et al., 1998). The expression data obtained by Northern analysis were confirmed by semi-quantitative RT-PCR as illustrated in Fig. 5B. A strongly fluorescent band of 1032 bp representing the coding region of LpCCR was observed in stem while no bands could be detected in root, leaf and flower. GAPDH expression was equally high in the tested tissues and thereby served as an internal control of the cDNA preparations used in RT-PCR experiments. The RT-PCR detects no CCR transcript in flowers while a very weak hybridisation signal is observed in Northern analysis which could be explained by unspecific hybridisation.

Example 4: Phylogeny

In an effort to determine the evolutionary relationships between members of the CCRs, a phylogenetic tree was generated using computer software (MEGALIGN program, Clustal method). A phylogenetic analysis (Fig. 6) of eight different plant CCRs has been carried out. As seen in the evolutionary tree of amino acid sequences, CCRs separate into three groups reflecting their evolution. One distinct group contains all monocot CCRs except maize CCR2, another group contains all the dicot CCRs, and the last group comprises the maize CCR2. The ryegrass CCR clearly belongs to the monocotyledonus clade including CCR from sugarcane, Festuca and maize (CCR1) and groups together with Fescue CCR. It is noteworthy that moncot and dicots CCRs fall into either side of the root and that the maize CCR2 seems to belong to a third distinct group of CCRs. The maize CCR2 gene has been isolated and analysed by Pichon et al. (1998). The gene exhibits a high sequence homology with ZmCCRI

(see also Fig. 2) and homology, although lower, to CAD1 and DFR. The amino acids comprising the catalytic site of CCR are not completely conserved in maize CCR2. The maize CCR2 cDNA was isolated from an iron deficient maize root library and might therefore be involved in stress responses. The divergation of monocotyledones and dicots have predated the duplication and specialization of plant CCRs. The dicot CCRs form a distinct group and the second maize CCR (CCR2) seems to even more evolutionary distant from the monocot group and closer to the dicot group. Lacombe et al. (1997) have performed a phylogenetic analysis of CCRs, dihydroflavone reductases and other reductases and found that these diffrent enzymes all share a common ancestor. Moreover, they suggest that

CCR can be considered as a new member of the mammalian 3β-hydroxysteroid dehydrogenase/plant dihydroflavone reductase superfamily. This is further supported by the matching positons of introns in LpCCR and dicot DFR genes. The positions of the four introns in LpCCR perfectly matches the positions of introns in plant DFR genes and Eucalyptus gunnii CCR. These results indicateds that gain or loss of the first intron (DFR) occured after a common ancestral species of ryegrass and eucalyptus had diverged from an ancestor.

Example 5: Transformation of ryegrass and fescue

Antisense and overexpression studies with the ryegrass CCR gene under the control of the ryegrass CCR promoter is carried out in a homologous system, i.e. in transgenic ryegrass plants. Alternatively, such experiments can also be performed with the closely related grass tall fescue (Festuca arundicinea) which often proves to be more ideal for transformation and regeneration. Direct gene transfer to protoplasts is one of several methods which have been developed for and applied for the generation of transgenic plants and for transient gene expresson studies, especially in monocots. The principle of the method is based on the efficient uptake of plasmid DNA from a cultivating medium which is promoted either by a chemical e.g. polyethylene glycol and/or by the application of electric pulses. The transformation procedure involves the prepation of protoplasts by enzymatic digestion, the addition of plasmid (DNA) harbouring the gene of interest to the protoplast suspension, uptake of DNA stimulated by chemicals and/or electroporation, selection, and finally, induced morphogenesis on developing calli leading to plant regeneration. Direct gene transfer to protoplast in Lolium has been reported in several cases (Potrykus et al., 1985; Murray et al., 1992; Wang et al., 1997). However, only circumstantial evidence for generation of transgenic ryegrass form protoplasts have been provided (Wang et al., 1997). Therefore another method for transformation of ryegraas can be applied i.e. protoplast-independent gene transfer techniques: the biolistic method. The biolistic method or paticle bombardment transformation, which overcomes limitations due to Agrobacterium- host specificity, involves gene delivery into intact plant cells and tissues by microprojectiles coated with biologically active DNA (Sanford et al., 1987; 1993; Sanford, 1988). In Lolium, both transient expression (Hensgens et al., 1993; Perez-

Vicente et al., 1993) transformed calli and transgenic plants (Spangenberg et al, 1995) have been generated using biolistics. Transgenic perrenial ryegrass (Lolium perenne) plants have been obtained by microprojectile bombardment of single genotype-erived embrygenic suspension cells (Spangenberg et al., 1995). Chimeric consctructs containing gusA genes under control of the CaMV35 promoter and maize ubiquitin promoter were used together with a chimeric act1-hph (hygromycin phosphotransferase) gene as selection marker.

Constructs: For the sense constructs, the complete sequence of SEQ ID NO 1 and SEQ ID NO 2 is inserted into the transformation vector. For the antisense construct, the antisense version of SEQ ID NO 2 is inserted under the control of SEQ ID NO 1. For each of these constructs a number of deletion mutants of the promoter sequence are made. The actual point where the sequence is cut can vary a few bases from the indications here below if the sequence is cut with a restriction enzyme. Otherwise, the sequences can be amplified using PCR. One deletion mutant containing nucleotides 430-1269 One deletion mutant containing nucleotides 780-1269 One deletion mutant containing nucleotides 940-1269 One deletion mutant containing nucleotides 1070-1269 One deletion mutant containing nucleotides 1-450 and 1070-1269 (deletion of nucleotides between 450 and 1070).

For each of the deletion mutants the expression level and the amount of lignin in stably transformed plants is measured.

Example 6: CCR antisense and sense experiments in Arabidopsis

Genes encoding cinnamoyl CoA reductase (CCR) have been isolated from various plants including both monocots and dicots. The amino acid sequences of the ryegrass CCR (this report) and the two CCR polypeptides predicted from the genes isolated from Arabidopsis thaliana (Lauvergeat et al., 2001) reveal a very high degree of identity which makes it very likely that an antisense construct of ryegrass CCR will work in this heterologous system. In order to evaluate the effects of down- regulating (antisense) and up-regulating the expression of CCR two constructs of the ryegrass CCR gene were generated. One containing the coding open reading frame of the ryegrass CCR, LpCCR, cDNA in sense orientation and another version with the open reading frame in antisense orientation. The ryegrass CCR 1035 bp coding element was inserted into the plant transformation vector pPZP211-GFP in either sense or antisense orientation. Linkers containing the restriction endonuclease sites X al and Sa HI were addded to the CCR gene by PCR. The plant transformation vector pPZP211-GFP (kindly provided by Dr. Erik østergaard Jensen, Laboratory for Gene Expression, University of Aarhus, Denmark) a modified version of the vector pPZP211 (Hajdukiewicz et al., 1994) was digested with SamHI and Xho\ resulting in the release of the GFP element. The coding sequence of the ryegrass CCR gene was ligated into this vector in both sense and antisense orientation.

A vector based on the CCR gene in sense and antisense orientation can be ligated into the vector by releasing the GFP element together with the 35S promoter through digestion with Hind\\\ and Xπol and subsesquent ligation into the empty vector of the CCR promoter linked to the coding sequence of ryegrass CCR in sense and antisense orientation.

The resulting vectors were transformed into E. coli XL-1 blue cells and plasmid DNA was prepared and used for sequencing of the border regions. The successful cloning of the constructs was confirmed by DNA sequencing. The plasmid DNA representing the two constructs with 35S promoter shown in Fig. 7 were transformed into Agrobacterium tumefasciens C1C58 together with the plasmids representing the two constructs with the ryegrass CCR promoter shown in Fig. 8.

Seeds of Arabidopsis thaliana var. Columbia were placed in 12 cm circular pots filled with soil and stored for two days at 4°C and subsequently transfereed to a growth chamber. Plants were grown for approximately 2 weeks in short days (10 hrs. light/14 hrs. dark). Conditions were then changed to long days (14 hrs. light/10 hrs. dark) for two weeks to induce bolting. Plants were grown to a stage at which bolts were 10-15 cm tall. The emerging bolts were then clipped of to encourage growth of multiple secondary bolts. Transformation ol Arabidopsis plants was carried out using the floral dip method described by Clough and Bent (1998). Transformation was performed 7-8 days after clipping by dipping the inflorescences in a supsension of Agrobacterium tumefasciens containing 0.01% Silwet and 0.01

Dg/liter benzylaminopurine. Seeds were harvested 18 days after transformation and stored for 2 days at 4°C. Seeds were spread on 9 x 9 cm plates containing MS- medium supplied with 100 mg/liter kanamycin. Positive transformants will be recognised by a bright green colour and extensive root growth while untransformed plants appear pale yellow and finally bleach out and with arrested or no root growth.

Kanamycin resistant seedlings will subsequently be subjected to PCR analyses using gene-specific primers for the ryegrass CCR sequence and Southern analysis in order to confirm the presence of the transgene. The phenotype of positive Arabidopsis transformants is compared to wild-type (untransformed) plants. Histochemical analyses is carried out in transformed plants and compared to wild- type plants in order to clarify changes in spatial distribution of lignin. Total lignin amount is determined in sense and antisense plants with 35S and ryegrass CCR promoter and compared to untransformed plants. In situ analyses combined with Northern analysis reveal any changes in the expression of the CCR enzyme in the heterologous system. The deletion mutants mentioned in example 5 are also transformed into Arabidopsis to evaluate the activity of the ryegrass CCR promoter in this plant. Instead of using the CCR cDNA from ryegrass in Arabidopsis, the cDNA for Arabidopsis CCR can be used.

Example 7, Transformation of conifers

Transformation of conifers was carried out using techniques described in WO 00/46382 (SILVAGEN). Several constructs including the ryegrass CCR promoter were obtained. These constructs include either genomic and cDNA CCR genes controlled by the ryegrass CCR promoter, as well as a pCCR-GUS construct to test the expression pattern of the CCR promoter. These and other constructs used in this example are listed below: A pGA482-based vector containing a CaMV 35S-cDNA CCR construct;

A pBIC20-based vector containing a genomic fragment containing both the CCR promoter and coding region;

A pB1121 -based vector containing a CaMV 35S-cDNA CCR construct.

A pB1101 -based vector containing a pCCR-GUS construct for xylem directed expression of GUS;

A pGA482-based vector containing a CCR-genomic CCR construct.

In the above constructs, whereas the ryegrass CCR promoter is used, the coding CCR sequence is preferably taken from a conifer in question. As a model species P. abies was chosen. The techniques are however generally applicable to conifers, including Pinus, Larix, Picea and Abies, for which somatic embryo regeneration techniques are state of the art.

Starting materials for such vectors are in common use for the construction of plant transformation vectors and are generally available around the world from various labs. The pBI-series is commercially available from Clontech. The pGA482 vector is described in 1987 Methods Enzymol 153: 292-305 and is widely used for plant transformation. The pBIC20is a binary cosmid vector described by Meyer et al. 1996, in Genome Mapping in Plants, ed. Paterson, A. H. (Landis Biochemical Press, Austin, TX). Construction of the pGA482 and pBIC20 based plasmids are detailed in Meyer et al., 1996, PNAS, 93.6869-6874 and both are available from that source (Chappie). The other constructs were made using similar techniques.

The CaMV 35S constructs have been used successfully to modify lignin content in both Arabidopsis and tobacco and were included in the present examples to give ectopic expression of the CCR gene in spruce and pine. The CCR promoter constructs should direct expression to the xylem, the principal target tissue for lignin modification.

To initiate transformation experiments, the plasmids were transformed into E. coli and were subsequently purified by CsCI gradient centrifugation. Each plasmid was checked by restriction digest to confirm its identity. Standard procedures were used for coating gold particles with the plasmids and for microprojectile bombardment of spruce somatic embryos. Regeneration of transformed spruce callus was done on a very low level of kanamycin (2-μg/ml) and embryo maturation was done using routine protocols for spruce and pine.

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Annex 1 Lolium brasilianum Nees

Lolium bromoides Huds.

Lolium aechicum Rouv. 40 Lolium canadense Bernh. ex Rouv.

Lolium aegyptiacum Bell, ex Rouv. Lolium canadense Michx. ex Roem. &

Lolium agreste Hort. ex Roem. & Schult. Schult.

Lolium album Steud. Lolium canariense Steud. Lolium ambiguum Desp. Lolium cechicum Opiz

Lolium annuum Bernh. 45 Lolium coelorachis Forst. ex Steud.

Lolium annuum L. Lolium complanatum Schrad.

Lolium annuum Lam. Lolium compositum Thuill.

Lolium appenninum Brouss. Lolium compressum Boiss. & Orph. ex Lolium arenarium Rouv. Nym.

Lolium aristatum (Willd.) Lag. 50 Lolium crassiculme K. H. Rechinger

Lolium aristatum Lag. ex Nym. Lolium cristatum Pers.

Lolium arundinaceum (Schreb.) Darbysh. Lolium cuneatum Nevski

Lolium arundinaceum (Schreb.) SJ. Lolium cuneatum Nevski Darbyshire Lolium cylindricum Aschers. & Graebn.

Lolium arvense Schrad. 55 Lolium cylindricum C. Koch

Lolium arvense With. Lolium decipiens Dum.

Lolium arvense With. Lolium distachyum Linn,

Lolium asperum Roth ex Kunth Lolium distachyum Willd. ex Steud. Lolium berteronianum Steud. Lolium dorei B. Boiv.

Lolium boucheanum Kunth 60 Lolium dorei B. Boivin

Lolium brasilianum Nees Lolium dorei var. laeve B. Boivin Lolium durum C. Koch 40 Lolium jechelianum Opiz

Lolium elegans Steud. Lolium latum Roth ex Steud.

Lolium elongatum Hort. ex Rouv. Lolium lepturoide Lojac.

Lolium felix Rouv. Lolium lepturoides Boiss. Lolium festuca Rasp, ex Mutel Lolium lesdaini Sennen

Lolium festuca Raspail 45 Lolium linicolum A. Br.

Lolium festucaceum Link Lolium loliaceum (Bory & Chaub.) Hand.-

Lolium festucoides Rasp, ex Mutel Mazz.

Lolium festucoides Raspail Lolium loliaceum Hand.-Mazz. Lolium flagellare Sprun. ex Boiss. Lolium longiglume St. Lager

Lolium gaudini Parl. 50 Lolium lowei Menezes

Lolium giganteum (L.) Darbysh. Lolium lucidum Dum.

Lolium giganteum (L.) S.J. Darbyshire Lolium macilentum Delastre

Lolium giganteum Hort. ex Roem. & Lolium marschallii Stev. Schult. Lolium maximum Guss.

Lolium glumosum Plan. 55 Lolium maximum Willd.

Lolium gmelini Lolium mazzettianum (E.B. Alexeev) S.J.

Lolium gracile (Dumort.) B.D. Jackson Darbyshire

Lolium gracile Dum. Lolium multiflorum forma submuticum Lolium gracile Hegetschw. (Mutel.) Anghel & Beldie

Lolium gracile Parl. 60 Lolium multiflorum Gaud.

Lolium grandispicum Y.J. Fei Lolium multiflorum Guss.

Lolium gussonei Nym. Lolium multiflorum Lam.

Lolium halleri C. C. Gmel. Lolium multiflorum Lam. Lolium hubbardii B.K.Simon Lolium multiflorum Lam.

Lolium hubbardii Jansen & Wachter 65 Lolium multiflorum Lam.

Lolium hubbardii Jansen & Wachter ex Lolium multiflorum var. typicum Stuck.

B.K. Simon Lolium oechicum Rouv.

Lolium humile Rouy Lolium osiridis Fig. & Delile ex Rouv. Lolium husnoti Sennen Lolium parabolicae Sennen

Lolium hybridum Hausskn. 70 Lolium perenne Aitch.

Lolium hybridum Hausskn. Lolium perenne forma anomala Hack.

Lolium infelix Rouv. Lolium perenne L.

Lolium italicum A. Br. Lolium perenne Linn. Lolium italicum A. Br. Lolium perenne subsp. rigidum (Gaud.) A.

Lolium italicum A. Br. 75 & D. Love

Lolium italicum A.Braun Lolium perenne var. brasilianum (Nees)

Lolium italicum var. muticum Doell in Kuntze

Mart. Lolium perenne var. cristatum Pers. Lolium perenne var. italicum (A. Br.) in Lolium subulatum Degen ex Lojac.

Parnell 40 Lolium subulatum Vis.

Lolium perenne var. italicum (A.Braun) Lolium subulatum Vis.

Rodway Lolium suffultum Sieber ex Huter Lolium perenne var. perenne L. Lolium temulentum Bert, ex Steud.

Lolium persicum Boiss. Lolium temulentum forma glaberrimum

Lolium persicum Boiss. & Hohen. 45 Kuntze

Lolium phoenice Rouv. Lolium temulentum forma scabrum (Koch)

Lolium phoenix Rouv. Soo Lolium pratense (Huds.) Darbysh. Lolium temulentum L.

Lolium pratense (Huds.) S.J. Darbyshire Lolium temulentum Linn.

Lolium pseudo-italicum Schur 50 Lolium temulentum subsp. arvense (With.)

Lolium pseudo-linicolum Gennari Tsvelev

Lolium remotum forma asperum (Roth) Lolium temulentum subsp. cuneatum Anghel & Beldie (Nevski) Tsvelev

Lolium remotum Schrank Lolium temulentum subsp. remorum

Lolium remotum Schrank 55 (Schrank) A. & D. Love

Lolium repens Lolium temulentum Thunb.

Lolium rigidum Gaud. Lolium temulentum var. arvense (With.) Lolium rigidum Gaudin Lilj.

Lolium rigidum subsp. negevense N. Lolium temulentum var. leptochaeton A.

Feinbrun-Dothan 60 Br.

Lolium rigidum var. aristatum Hack. Lolium temulentum var. linicola Benth.

Lolium rigidum Weiss ex Nym. Lolium temulentum var. macrochaeton A. Lolium robustum Reichb. Br.

Lolium romanum Sang. Lolium temulentum var. multiflorum

Lolium rosetlanum Fig. & Delile ex Rouv. 65 Kuntze

Lolium scabrum J. & C. Presl Lolium temulentum var. temulentum L.

Lolium sect. Eulolium Godr. Lolium tenue Bieb. Lolium siculum Parl. Lolium tenue Guss.

Lolium sonderi Rouv. Lolium tenue Linn.

Lolium speciosum Stev. ex Bieb. 70 Lolium teres H. Lindb.

Lolium strictum Decker ex Steud. Lolium trabuti Hochr.

Lolium strictum Presl Lolium triticoides Janka Lolium subgen. Schedonorus (P. Beauv.) Lolium vulgare Host

Darbysh. Lolium xerophilum Domin

Lolium subgen. Schedonorus (P. Beauv.)75

S.J. Darbyshire

Claims

1. A regulatory polynucleotide capable of promoting the expression of a coding polynucleotide sequence linked to its 3' end, selected from the group comprising:

A) the 1269 nucleotide sequence of SEQ ID NO 1 ;

B) 1269 nucleotides of the 1333 nucleotide sequence contained in plasmid pLPCCR deposited with the DSMZ under accession number DSM 14003;

C) a fragment comprising at least 100 contiguous nucleotides of the sequence of SEQ ID NO 1 ;

D) a functional variant having at least 65% sequence identity to SEQ ID NO 1 ;

E) a functional variant of the fragment of item C) having at least 80 % sequence identity to the fragment;

F) a polynucleotide having a 5' end and a 3' end defined respectively by a transcription start site of a Lolium perenne CCR and a Sa/I restriction site located 5' to the transcription start site;

G) a nucleotide sequence comprising at least 100 bases capable of hybridising to any one of A), B), C), D), E), or F) under conditions of under conditions of high stringency; and

H) the complementary polynucleotide sequence to any of the sequences of items A through G.

2. A polynucleotide comprising the 1269 nucleotide sequence of SEQ ID NO 1

3. A polynucleotide comprising 1269 nucleotides of the 1333 nucleotide sequence contained in plasmid pLPCCR deposited with the DSMZ under accession number DSM 14003.

4. The polynucleotide according to claim 2 or 3, comprising a fragment comprising at least 100 contiguous nucleotides of the sequence of SEQ ID NO 1.

5. A polynucleotide according to claim 2 or 3, comprising a functional variant having at least 65% sequence identity to the sequences of said claims.

6. A polynucleotide according to claim 4, comprising a functional variant having at least 80 % sequence identify to the sequences of said claim.

7. A polynucleotide having a 5' end and a 3' end defined respectively by a transcription start site of a Lolium perenne CCR and a Sa/I restriction site located 5' to the transcription start site.

8. A polynucleotide comprising a nucleotide sequence comprising at least 100 bases capable of hybridising to a polynucleotide according to any of claims 2 to 7 of under conditions of high stringency.

9. A polynucleotide being complementary to any of the sequences of any of claims 2 to 8.

10. The polynucleotide according to any of claims 1 to 8, wherein a fragment of SEQ ID NO 1 comprises at least nucleotides no 1-1250, such as at least no 1-1230, for example at least no 1-1210, such as at least 1-1190, for example at least 1- 1170, such as from 1-1150, for example at least 1-1130, such as at least 1-

1110, for example at least 1-1090, such as at least 1-1070, for example at least 1-1050, such as at least 1-1030, for example at least 1-1010, such as at least 1- 990, such as at least 1-970, for example at least 1-950, such as at least 1-930, for example at least 1-910, such as at least 1-890, for example at least 1-870, such as at least 1-850, such as at least 1-830, for example at least 1-810, such as at least 1-790, for example at least 1-770, such as at least 1-750, for example at least 1-730, such as from 1-710, for example at least 1-690, such as at least 1-670, for example at least 1-650, such as at least 1-630, for example at least 1- 610, such as at least 1-590, such as at least 1-570, for example at least 1-550, such as at least 1-530, for example at least 1-510, such as at least 1-490, for example at least 1-470, such as at least 1-450, for example at least 1-430, such as at least 1-410, for example at least 1-390, such as at least 1-370, for example at least 1-350, such as at least 1-330, for example at least 1-310, such as at least 1-290, for example at least 1-270, such as at least 1-250, for example at least 1-230, such as at least 1-210, for example at least 1-190.

11. The polynucleotide according to any of claims 1 to 8, wherein a fragment of SEQ ID NO 1 comprises at least nucleotides no 20-1269, such as at least no 40 to 1269, for example at least 60 to 1269, such as at least 80-1269, for example at least 100 to 1269, such as at least 120 to 1269, for example at least 140-1269, such as at least 160-1269, for example at least 160-1269, such as at least 180- 1269, such as at least 200-1269, for example at least 220-1269, such as at least 240-1269, for example at least 260-1269, such as at least 280-1269, such as at least 300-1269, for example at least 320-1269, such as at least 340-1269, such as at least 360-1269, for example at least 380-1269, such as at least 400-1269, for example at least 420-1269, such as at least 440-1269, such as at least 460- 1269, for example at least 480-1269, such as at least 500-1269, for example at least 520-1269, such as at least 540-1269, for example at least 560-1269, such as at least 580-1269, for example at least 600-1269, such as at least 620-1269, for example at least 640-1269, such as at least 660-1269, for example at least

680-1269, such as at least 700-1269, for example at least 720-1269, such as at least 740-1269, for example at least 760-1269, such as at least 780-1269, for example at least 800-1269, such as at least 820-1269, for example at least 840- 1269, such as at least 860-1269, for example at least 880-1269, such as at least 900-1269, for example at least 920-1269, such as at least 940-1269, for example at least 960-1269, such as at least 980-1269, for example at least 1000-1269, such as at least 1020-1269, for example at least 1040-1269, such as at least 1060-1269, for example at least 1080-1269, such as at least 1100-1269, for example at least 1120-1269, such as at least 1140-1269, for example at least 1160-1269.

12. The polynucleotide according to any of claims 1 to 8, wherein a fragment of SEQ ID NO 1 comprises at least one fragment selected from the group consisting of nucleotides 1 to 100, 101 to 200, 201 to 300, 301 to 400, 401-500, 501-600, 601-700, 701-800, 801-900, 901-1000, 1001 -1100, 1101 -1200, 1101 -1269.

13. The polynucleotide according to any of claims 1 to 8, comprising at least one regulatory sequence selected from the group of sequences consisting of: i) a CAAT sequence; ii) a ACCTAACT sequence showing homology to box P in promoters of phenylpropanoid biosynthesis genes and to a MYB consensus binding site; iii) a TGGTAAAG sequence comprising a prolamin binding box, which binds a P-box binding factor; iv) a GGATA-motif being a core motif of MybSta binding site; v) a CCAACC motif comprising a core of a MYB homologue binding site; vi) a GGTCAGAGGG sequence; vii) a GCAGAGTACTC sequence; viii) a CGATCCTCA sequence; ix) a CCAACAAG sequence; x) a CCAATCA sequence; xi) a GCCAGTTT sequence; xii) a GTAAAAGA sequence; xiii) a CAAAGAA sequence; xiv) a TGTAGCT sequence; xv) a GGAAC sequence.

14. The polynucleotide according to claim 12 or 13, comprising at least a transcription start site and at least one regulatory sequence, such as at least two regulatory sequences, for example at least 3 regulatory sequences, such as at least 4 regulatory sequences, for example at least 5 regulatory sequences, such as at least 8 regulatory sequences, for example at least 10 regulatory sequences, such as at least 15 regulatory sequences, for example at least 20 regulatory sequences.

15. The polynucleotide sequence according to claim 14, comprising at least fragments i), ii), iii), and iv) of claim 13.

16. The polynucleotide according to claim 1 further comprising at least 20 bases located 5' to nucleotide number 1 of SEQ ID NO 1 in Lolium perenne var. Borvi, such as at least 40 bases, for example at least 60 bases, such as at least 80 bases, for example at least 100 bases, such as at least 150 bases, for example at least 200 bases, such as at least 250 bases, for example at least 350 bases, such as at least 500 bases, for example at least 750 bases, such as at least 1000 bases, for example at least 1250 bases, such as at least 1500 bases.

17. The polynucleotide according to claim 1 , further comprising at least 20 bases located 5' to the Sa/I restriction site 5' to the translation start codon of the CCR gene in Lolium perenne, such as at least 40 bases, for example at least 60 bases, such as at least 80 bases, for example at least 100 bases, such as at least 150 bases, for example at least 200 bases, such as at least 250 bases, for example at least 350 bases, such as at least 500 bases, for example at least 750 bases, such as at least 1000 bases, for example at least 1250 bases, such as at least 1500 bases.

18. The polynucleotide according to any of the preceding claims, wherein the expression is tissue specific.

19. The polynucleotide according to claim 18, wherein the number of transcripts produced in one tissue type is at least 10 times higher than in another tissue type, more preferably at least 100 times higher, such as at least 1000 times higher, for example at least 10,000 times higher, such as at least 100,000 times higher, for example at least 1 ,000,000 times higher

20. The polynucleotide according to claim 18 or 19, wherein expression is confined to lignin producing tissue.

21. The polynucleotide according to claim 18 or 19, wherein the tissue comprises stem tissue.

22. A CCR gene, which comprises in it's 5' non-coding region an expression signal sequence of at least 1.2 kb, wherein the promoter sequence comprises: a TATAAAT-sequence ( ATA-box') at position 1182-1188; a transcription starting site downstream from the TATA-box; a startcodon at position 1334-1336; and at least one of the following regulatory sequences: i) a CAAT sequence at position 958-961 ; ii) a ACCTAACT sequence at position 1081-1088 showing homology to box P in promoters of phenylpropanoid biosynthesis genes and to a MYB consensus binding site; iii) a TGGTAAAG sequence at position 938-945 comprising a prolamin binding box, which binds a P-box binding factor; iv) a GGATA-motif at position 439-443 being a core motif of MybSta binding site; v) a CCAACC motif at position 72-78 comprising a core of a MYB homologue binding site; vi) a GGTCAGAGGG sequence at position 910 to 919; vii) a GCAGAGTACTC sequence at position 845-855; viii) a CGATCCTCA sequence at position 697-705; ix) a CCAACAAG sequence at position 582-589; x) a CCAATCA sequence at position 957-963; xi) a GCCAGTTT sequence at position 1014-1021 ; xii) a GTAAAAGA sequence at position 820-828; xiii) a CAAAGAA sequence at position 789-795; xiv) a TGTAGCT sequence at position 561-567; xv) a GGAAC sequence at position 398-402.

23. The CCR gene according to claim 22, comprising at least the regulatory sequences i), ii), iii), and iv).

24. The CCR gene according to claims 22 or 23, being isolated from an angiosperm, preferably from a monocot, more preferably from a Gramineae, more preferably from a Festuceae, more preferably from a Lolium spp, more preferably from a Lolium perenne.

25. A nucleotide construct comprising a promoter region comprising at least a regulatory polynucleotide according to claims 1 to 21 and a coding region operably linked to said polynucleotide wherein the expression of said coding region changes the phenotype of a plant transformed with said nucleotide construct.

26. The construct according to claim 25, further comprising a 5' untranslated sequence between the coding sequence and the regulatory polynucleotide.

27. The construct according to claim 25, wherein the coding region comprises a gene in the lignin biosynthesis pathway.

28. The construct according to claim 27, wherein the gene comprises a gene selected from the group comprising: PAL, and/or C4H, and/or C3H, and/or OMT, and/or F5H, and/or 4CL, and/or CCR, and/or CAD, and/or pCCoASH, and/or CCoAOMT.

29. The construct according to claim 27, wherein the gene comprises a CCR gene and/or a CAD gene.

30. The construct according to claim 29, wherein the CCR gene comprises a CCR gene from a plant, such as from an angiosperm, such as from a monocot, such as from a grass, such as from a Lolium spp, such as from Lolium perenne.

31. The construct according to claim 30, wherein the CCR gene comprises SEQ ID NO 2.

32. The construct according to claim 30, wherein the CCR gene comprises a sequence coding for SEQ ID NO 3 or for one of SEQ ID NOs 5-11.

33. The construct according to claim 25, wherein the coding region comprises at least part of an anti-sense equivalent of a gene in the lignin biosynthesis pathway, such as an anti-sense equivalent of PAL, and/or C4H, and/or C3H, and/or OMT, and/or F5H, and/or 4CL, and/or CCR, and/or CAD, and/or pCCoASH, and/or CCoAOMT, more preferably an anti-sense equivalent of CCR and/or CAD.

34. The construct according to claim 33, wherein the antisense equivalent is capable of reducing the amount of functional gene product by at least 5 %, such as at least 10 %, for example at least 15%, such as at least 20%, such as at least 25%, for example at least 30%, such as at least 40%, such as at least 50%, for example at least 60%, such as at least 70%, for example at least 80%, such as at least 90%, for example at least 95%, such as at least 99%, for example substantially 100%.

35. The construct according to claim 33 or 34, comprising at least part of the anti- sense equivalent of SEQ ID NO 2.

36. The construct according to claim 33 or 34, comprising at least part of an anti- sense equivalent of a nucleotide sequence coding for SEQ ID NO 3 or for one of SEQ ID NO 5 to 1 1.

37. The construct according to claim 25, wherein the coding region comprises a reporter gene, such as GUS, GFP, luciferase, or anthocyanin genes.

38. The construct according to claim 25, wherein the coding region comprises a resistance gene.

39. The construct according to any of the preceding claims 25 to 38, further comprising a nucleotide sequence coding for a selectable marker.

40. The construct according to claim 39, wherein the selectable marker is selected from the group consisting of markers conferring resistance to antibiotics such as ampicillin, geneticin, hygromycin, tetracyclin, kanamycin, methotrexat G418, neomycin, or to herbicides such as glyphosate, or bialaphos.

41. The construct according to claim 39, wherein the selectable marker is selected from the group consisting of metabolic markers, such as a marker conferring the ability to metabolise unusual and/or toxic substrates such as 2-deoxyglucose, mannose.

42. The construct according to any of the preceding claims 25 to 41 , further comprising enhancers such as viral enhancers, such as the SV40 enhancer region, the 35S enhancer element, and the like.

43. The construct according to any of the preceding claims 25 to 42, further comprising a terminator sequence capable of functioning in a plant cell.

44. The construct according to any of the preceding claims 25 to 43, further comprising a spacer sequence between the promoter region and the coding sequence and/or at least one intron in the coding sequence.

45. A cloning vector comprising a nucleotide construct according to claims 25 to 44.

46. A transformation vector comprising a nucleotide construct according to claims 25 to 44.

47. A shuttle vector comprising a nucleotide construct according to claims 25 to 44 capable of stable replication in both E. coli, A. tumefaciens, and Agrobacterium rhizogenes.

48. A host cell comprising a nucleotide construct according to claims 25 to 44.

49. A host cell comprising a vector according to claims 45 to 47.

50. A host cell according to claims 48 or 49 comprising a plant cell, selected from the group comprising pollen cells, callus cells, tissue cultures, cell cultures, protoplast cultures, plant protoplasts, plant organs, plant tissues, seeds, embryos, egg-cells, zygotes.

51. A host cell according to claims 48 or 49, comprising a yeast cell.

52. A host cell according to claims 48 or 49, comprising a bacterial cell, selected from the group comprising E. coli, Bacillus subtilis.

53. A transgenic plant comprising a construct according to claims 25 to 44.

54. The plant according to claim 53, wherein the plant comprises an angiosperm, such as a monocot or a dicot.

55. The plant according to claim 54, wherein the monocot comprises a species selected from the group comprising the family Gramineae, such as the subfamily Festucoideae, such as the Festuceae tribe, such as the Lolium, Festuca Dactylis, Poa, Bromus, or Brachypodium genera.

56. The plant according to claim 55, wherein the Lolium sp. comprises Lolium perenne L., Lolium multiflorum L., Lolium x boucheanum Kunth.

57. The plant according to claim 55, wherein the Festuca sp. comprises Festuca arundinaceae L., Festuca pratensis Huds., Festuca rubra L., Festuca ovina,

Festuca ovina ssp. duriuscula,

58. The plant according to claim 55, wherein the species comprises a species selected from the group comprising Dactylis glomerata L., Poa trivialis L., Poa palustris L., Poa pratensis L., Bromus catharticus, Bromus sitchensis,

Miscanthus spp.

59. The plant according to claim 54, comprising a woody species such as a Populus spp, an Eucalyptus spp, a Hevea spp, such as Hevea brasiliensis, a Quercus spp, a Betula spp, a Fagus spp, a Fraxinus spp, an Ulmus spp, a Liriodendron spp, a Liquidambar spp, a Robinia spp.

60. The plant according to claim 53, comprising a gymnosperm, such as a conifer, such as a Pinus spp, for example P. taeda, P. radiata, P. silvestris, P. lambertiana, P. elliotti, P. palustris; a Picea spp such as P. abies, P. rubra, P. stichensis, P. rubra, P. mariana, P. glauca; a Larix spp, such as L. leptolepis, L. decidua, L. occidentalis, L. laricina; an Abies spp. such as A. grandis, A. fraserii, A. concolor, A. procera, A. balsamea; a Pseudotsuga spp such as P. menziesii; a Tsuga spp; a Thuja spp; a Chamacyparis spp.; a Sequoia sppp.

61. Propagation material from a plant according to claims 53 to 60.

62. Propagation material according to claim 61 , being selected from the group comprising seeds, cuttings, cell cultures, tissue cultures, zygotes, pollen, egg- cells, somatic embryos, artificial seeds, adventitious buds, axillary buds, grafts.

63. Part of a plant according to claims 53 to 60.

64. A method for preparation of a polynucleotide sequence according to claims 1-21 comprising i) chemical synthesis of the sequences, or ii) cloning of the sequences from the 5' non transcibed region of a plant CCR gene.

65. The method according to claim 64, whereby the sequences are cloned from Lolium perenne CCR.

66. The method according to claims 64 or 65, comprising the steps of i) extraction and purification of genomic DNA from tissues capable of synthesising lignin, ii) fragmentation of the isolated DNA using at least one restriction enzyme, iii) cloning of fragments of suitable size into a cloning vector obtaining a gene library, iv) identification of clones comprising a CCR gene or a part of a CCR gene using a CCR specific probe, v) isolation and sequencing of the identified clones.

67. The method according to any of claims 64-66, further comprising modification of the sequences by site directed mutagenesis and/or PCR-priming obtaining functional variants and/or cutting with restriction enzymes.

68. A method for preparation of a nucleotide construct comprising insertion of a promoter region comprising a polynucleotide sequence according to claims 1 to 21 immediately before the start codon of a desired coding sequence.

69. The method according to claim 68, further comprising insertion of a spacer sequence between the promoter region and the start codon.

70. The method according to claim 68 or 69, wherein the coding sequence comprises a coding sequence as defined by claims 25-44.

71. A method for preparation of an expression vector comprising a coding nucleotide sequence capable of being expressed in a plant cell operably linked to a promoter region comprising a polynucleotide according to claims 1 to 21 , comprising i) insertion of said promoter region immediately before the start codon of said coding nucleotide sequence, obtaining a construct, ii) operably inserting said construct into a plant expression vector, the expression vector further comprising a selectable marker.

72. A method according to claim 71 , further comprising insertion of the construct between further expression signals, such as a further promoter, enhancer or repressor sequence and/or a terminator sequence.

73. A method for controlling the amount of lignin in a plant comprising the steps of: i) providing a construct comprising a non-coding regulatory polynucleotide according to claims 1 to 21 and a coding region comprising a sequence coding for an enzyme in the lignin biosynthesis pathway in sense or anti- sense orientation, ii) inserting the construct into a plant cell, iii) obtaining plants with an increased, with a decreased amount of lignin or with an altered lignin composition.

74. The method according to claim 73, whereby step ii) comprises the use of Agrobacterium tumefaciens, A. rhizogenes, protoplast transformation such as

PEG-mediated gene uptake, particle bombardment, whiskers mediated transformation, pollen mediated transformation, electroporation, vacuum infiltration.

75. A method for identifying new regulatory polynucleotide sequences comprising the use of an isolated polynucleotide or a fragment of a polynucleotide according to claims 1 to 13 as a probe for identification of homologous DNA sequences by screening of cDNA or genomic libraries.