AU3639900A - A method for regulation of plant lignin composition - Google Patents

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S&F Ref: 428367D1

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

Name and Address of Applicant: a Actual Inventor(s): Address for Service: Invention Title: Purdue Research Foundation Office of Technology Transfer 1063 Hovde Hall West Lafayette Indiana 47907 United States of America Clint Chapple Spruson Ferguson St Martins Tower 31 Market Street Sydney NSW 2000 A Method for Regulation of Plant Lignin Composition The following statement is a full description of this invention, including the best method of performing it known to me/us:- IP Australia Documents received on: o 2 4 MAY 2000 Batch No: 5845c a

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TITLE

A METHOD FOR REGULATION OF PLANT LIGNTN COMPOSITION FIELD OF INVENTION The present method relates to the field of molecular biology and the regulation of protein synthesis through the introduction of foreign genes into plant genomes. More specifically, the method relates to the modification of plant lignin composition in a plant cell by the introduction of a foreign plant gene encoding an active ferulate-5-hydroxylase (F5H) enzyme. Plant transformants harborifig the gene demonstrate increased levels of syringyl monomer residues in their lignin, a trait that is thought to render the polymer more susceptible to delignification.

BACKGROUND

Lignin is one of the major products of the general phenylpropanoid pathway, and is one of the most abundant organic molecules in the biosphere (Crawford, (1981) Lignin Biodegradation and Transformation, New York: John Wiley and Sons). In nature, lignification provides rigidity to wood and is in large part responsible for the structural integrity of plant tracheary elements. Lignin is well suited to these capacities because of its physical characteristics and its resistance to biochemical degradation. Unfortunately, this same resistance to 20 degradation has a significant impact on the utilization of lignocellulosic plant material (Whetten et al., Forest Ecol. Management 43, 301, (1991)).

The monomeric composition of lignin has significant effects on its chemical degradation during industrial pulping (Chiang et al., Tappi, 71, 173, (1988). The guaiacyl lignins (derived from ferulic acid) characteristic of softwoods such as pine, 25 require substantially more alkali and longer incubations during pulping in comparison to the guaiacyl-syringyl lignins (derived from ferulic acid and sinapic acid) found in hardwoods such as oak. The reasons for the differences between these two lignin types has been explored by measuring the degradation of model compounds such as guaiacylglycerol-P-guaiacyl ether, syringylglycerol-P-guaiacyl 30 ether, and syringylglycerol-0-(4-methylsyringyl) ether (Kondo et al., Holzforschung, 41, 83, (1987)) under conditions that mimic those used in the pulping process. In these experiments, the mono- and especially di-syringyl compounds were cleaved three to fifteen times faster than their corresponding diguaiacyl homologues. These model studies are in agreement with studies comparing the pulping of Douglas fir and sweetgum wood where the major differences in the rate of pulping occurred above 150 'C where arylglycerol-P-aryl ether linkages were cleaved (Chiang et al., Hol=forschung, 44, 309, (1990)).

Another factor affecting chemical degradation of the two lignin forms may be the condensation of lignin-derived guaiacyl and syringyl residues to form diphenylmethane units. The presence of syringyl residues in hardwood lignins leads to the formation of syringyl-containing diphenylmethane derivatives that remain soluble during pulping, while the diphenylmethane units produced during softwood pulping are alkali-insoluble and thus remain associated with the cellulosic products (Chiang et al., Holzforschung, 44, 147, (1990); Chiang et al., Holzforschung, 44, 309, (1990)). Further, it is thought that the abundance of crosslinks that can occur between guaiacyl residues contributes to resistance to chemical degradation. This linkage is resistant to alkali cleavage and is much less common in lignin that is rich in syringyl residues because of the presence of the 5-O-methyl group in syringyl residues. The incorporation of syringyl residues results in what is known as "non-condensed lignin", a material that is significantly easier to pulp than condensed lignin.

Similarly, lignin composition and content in grasses is a major factor in determining the digestibility of lignocellulosic materials that are fed to livestock (Jung, H.G. Deetz, D.A. (1993) Cell wall lignification and degradability in Forage Cell Wall Structure and Digestibility Jung, D.R Buxton, R.D.

Hatfield, and J. Ralph eds.), ASA/CSSA/SSSA Press, Madison, The 20 incorporation of the lignin polymer into the plant cell wall prevents microbial enzymes from having access to the cell wall polysaccharides that make up the wall.

As a result, these polysaccharides cannot be degraded and much of the valuable carbohydrates contained within animal feedstocks pass through the animals Sundigested. Thus, an increase in the dry matter of grasses over the growing season is counteracted by a decrease in digestibility caused principally by increased cell wall lignification. From these examples, it is clear that the modification of lignin monomer composition would be economically advantageous.

The problem to be overcome, therefore, is to develop a method for the creation of plants with increased levels of syringyl residues in their lignin to 30 facilitate its chemical degradation. Modification of the enzyme pathway responsible for the production of lignin monomers provides one possible route to solving this problem.

The mechanism(s) by which plants control lignin monomer composition has been the subject of much speculation. As mentioned earlier, gymnosperms do not synthesize appreciable amounts of syringyl lignin. In angiosperms, syringyl lignin deposition is developmentally regulated: primary xylem contains guaiacyl lignin, while the lignin of secondary xylem and sclerenchyma is guaiacyl-syringyl lignin (Venverloo. Holzforschung 25, 18 (1971); Chapple et al., Plant Cell 4, 1413, a (1992)). No plants have been found to contain purely syringyl lignin. It is still riot clear how this specificity is controlled; however, at least five possible enzymatic control sites exist, namely caffeic acid/5-hydroxyferulic acid O-methyltransferase (OMT), F5H, (hydroxy)cinnamoyl-CoA ligase (4CL), (hydroxy)cinnamoyl-CoA reductase (CCR), and (hydroxy)cinnamoyl alcohol dehydrogenase (CAD). For example, the substrate specificities of OMT (Shimada et al., Phytochemistry, 22, 2657, (1972); Shimada et al., Phytochemistry, 12, 2873, (1973); Gowri et aL, Plant Physiol., 97, 7, (1991); Bugos et al., Plant Mol. Biol. 17, 1203, (1992)) and CAD (Sarni et al., Eur. J. Biochem., 139, 259, (1984); Goffner et al., Planta., 188, 48, (1992); O'Malley et al., Plant Physiol., 98, 1364, (1992)) are correlated with the differences in lignin monomer composition seen in gymnosperms and angiosperms, and the expression of 4CL isozymes (Grand et al., Physiol. Veg. 17, 433, (1979); Grand et al., Planta., 158, 225, (1983)) has been suggested to be related to the tissue specificity oflignin monomer composition seen in angiosperms.

Although there are at least five possible enzyme targets that could be exploited, only OMT and CAD have been investigated in recent attempts to manipulate lignin monomer composition in transgenic plants (Dwivedi et al., Plant Mol. Biol. 26, 61, (1994); Halpin et al., Plant J. 6, 339, (1994); Ni et al., 20 Transgen. Res. 3, 120 (1994); Atanassova et al., Plant J. 8, 465, (1995); Doorsselaere et al., Plant J. 8, 855, (1995)). Most of these studies have focused on sense and antisense suppression of OMT expression. This approach has met with variable results, probably owing to the degree of OMT suppression achieved in the various studies. The most dramatic effects were seen by using homologous OMT constructs to suppress OMT expression in tobacco (Atanassova et al., supra) and poplar (Doorsselaere et al., supra). Both of these studies found that as a result of transgene expression, there was a decrease in the content of syringyl lignin and a concomitant appearance of 5-hydroxyguaiacyl residues. As a result of these studies, Doorsselaere et al., (WO 9305160) disclose a method for the regulation of lignin biosynthesis through the genomic incorporation of an OMT gene in either the sense or anti-sense orientation. In contrast, Dixon et al.

(WO 9423044) demonstrate the reduction of lignin content in plants transformed with an OMT gene, rather than a change in lignin monomer composition. Similar research has focused on the suppression of CAD expression. The conversion of coniferaldehyde and sinapaldehyde to their corresponding alcohols in transgenic tobacco plants has been modified with the incorporation of an A. cordata CAD gene in anti-sense orientation (Hibino et al., Biosci. Biotechnol. Biochem., 59, 929, (1995)). A similar effort aimed at antisense inhibition of CAD expression l 1, generated a lignin with increased aldehyde content, but only a modest change in lignin monomer composition (Halpin et al., supra). This research has resulted in the disclosure of methods for the reduction of CAD activity using sense and antisense expression of a cloned CAD gene to effect inhibition of endogenous CAD expression in tobacco [Boudet et al., 5,451,514) and Walter et al., (WO 9324638); Bridges et al., (CA 2005597)]. None of these strategies increased the syringyl content of lignin, a trait that is correlated with improved digestibility and chemical degradability of lignocellulosic material (Chiang et al., supra; Chiang and Funaoka, Holzforschung 44, 309 (1990); Jung et al., supra).

Although F5H is also a key enzyme in the biosynthesis of syringyl lignin monomers it has not been exploited to date in efforts to engineer lignin quality. In fact, since the time of its discovery over 30 years ago (Higuchi et al., Can. J.

Biochem. Physiol., 41, 613, (1963)) there has been only one demonstration of the activity of F5H published (Grand, FEBSLett. 169, 7, (1984)). Grand demonstrated that F5H from poplar was a cytochrome P450-dependent monooxygenase (P450) as analyzed by the classical criteria of dependence on NADPH and light-reversible inhibition by carbon monoxide. Grand further demonstrated that F5H is associated with the endoplasmic reticulum of the cell.

The lack of attention given to F5H in recent years may be attributed in general to 20 the difficulties associated with dealing with membrane-bound enzymes, and specifically to the lability of F5H when treated with the detergents necessary for solubilization (Grand, supra). The most recent discovery surrounding the gene has been made by Chapple et al., (supra) who reported a mutant of Arabidopsis thaliana L. Heynh namedfahl that is deficient in the accumulation of sinapic acid-derived metabolites, including the guaiacyl-syringyl lignin typical of angiosperms. This locus, termed FAHI, encodes F5H. The cloning of the gene encoding F5H would provide the opportunity to test the hypothesis that F5H is a useful target for the engineering of lignin monomer composition.

In spite of sparse information about F5H in the published literature, 30 Applicant has been successful in the isolation, cloning, and sequencing of the gene. Applicant has also demonstrated that the stable integration of the F5H gene into the plant genome, where the expression of the F5H gene is under the control of a promoter other than the gene's endogenous promoter, leads to an altered regulation of lignin biosynthesis.

SUMMARY OF THE INVENTION The present invention provides isolated nucleic-acid fragments comprising the nucleotide sequences which correspond to SEQ ID NO.:1 and SEQ ID NO.:3 encoding an active plant F5H enzyme wherein the enzyme has the amino acid sequence encoded by the mature functional protein which corresponds to SEQ ID NO. :2 and wherein the amino acid sequence encompasses amino acid substitutions, additions and deletions that do not alter the function of the F5H enzyme.

The invention further provides a chimeric gene causing altered guaiacyl:syringyl lignin monomer ratios in a transformed plant, the gene comprising a nucleic acid fragment encoding an active plant F5H enzyme operably linked in either sense or antisense orientation to suitable regulatory sequences. The nucleic acid fragments are those described above.

Also provided is a method of altering the activity of F5H in a plant by means of transforming plant cells in a whole plant with a chimeric gene causing altered guaiacyl:syringyl lignin monomer ratios in a transformed plant cell, wherein the gene is expressed; growing said plants under conditions that permit seed development; and screening the plants derived from these transformed seeds for those that express an active F5H gene or fragment thereof.

A method is propvided of altering the activity of F5H enzyme in a plant by transforming a cell, tissue or organ from a suitable host plant with the chimeric gene desribed above wherein the chimeric gene is expressed; (ii) selecting transformed cells, cell callus, somatic embryos, or seeds which contain the chimeric gene; (iii) regenerating whole plants from the transformed cells, cell callus, somatic embryos, or seeds selected in step (iv) selecting whole plants regenerated in step (iii) which have a phenotype characterized by an ability of the whole plant to accumulate compounds derived from sinapic acid or an altered syringyl lignin monomer content relative to an untransformed host plant.

The invention additionally provides a method of altering the composition of 25 lignin in a plant by means of stably incorporating into the genome of the host plant by transformation a chimeric gene causing altered guaiacyl:syringyl lignin monomer ratios in a transformed plant; expressing the incorporated gene such that F5H is expressed and wherein guaiacyl:syringyl lignin monomer ratios are altered from those ratios of the untransformed host plant.

BRIEF DESCRIPTION OF THE FIGURES AN SEQUENCE LISTING Figure 1 illustrates the biosynthesis of monomeric lignin precursors via the general phenylpropanoid pathway.

Figure 2 is an illustration of the pBIC20-F5H cosmid and the overexpression construct (pGA482-35S-F5H) in which the F5H gene is expressed under the control of the constitutive cauliflower mosaic virus 35S promoter.

Figure 3 shows an analysis of sinapic acid-derived secondary metabolites in wild type, thefahl-2 mutant, and independently-derived transgenicfahl-2 plants carrying the T-DNA derived from the pBIC20-F5H cosmid, or the pGA482-35S-F5H overexpression construct.

Figure 4 shows the impact of F5H overexpression by comparing the steady state levels of F5H mRNA in wild type, thefahl-2 mutant, and independentlyderived transgenicfahl-2 plants carrying the T-DNA derived from the 35S-F5H overexpression construct.

Figure 5 shows a GC analysis of lignin nitrobenzene oxidation products toillustrate the impact of F5H overexpression on lignin monomer composition in the wild type, thefahl-2 mutant, and afahl-2 mutant carrying the T-DNA derived from the 35S-F5H overexpression construct.

Figure 6 illustrates a Southern blot analysis comparing hybridization of the cDNA to EcoRI digested genomic DNA isolated from wild type Arabidopsis thaliana and a number offahl mutants.

Figure 7 is a Northern blot analysis comparing hybridization of the cDNA to RNA isolated from wild type Arabidopsis thaliana and a number offahl mutants.

Figure 8 shows the genomic nucleotide (SEQ ID NO. and amino acid (SEQ ID NO.:2) sequences of the Arabidopsis F5H gene and the F5H enzyme that it encodes.

20 Applicant(s) have provided three sequence listings in conformity with 37 C.F.R. 1.821-1.825 and Appendices A and B ("Requirements for Application Disclosures Containing Nucleotides and/or Amino Acid Sequences") and in conformity with "Rules for the Standard Representation of Nucleotide and Amino Acid Sequences in Patent Applications" and Annexes I and II to the Decision of the President of the EPO, published in Supplement No 2. to OJ EPO, 12/1992.

The sequence of the Arabidopsis thaliana F5H cDNA is given in SEQ ID NO.:1 and the sequence of the Arabidopsis thaliana F5H genomic clone is given in SEQ ID NO.:3. The sequence of the F5H protein is given in SEQ ID NO.:2.

DETAILED DESCRIPTION OF THE INVENTION 30 The present invention provides a gene that encodes F5H, a key enzyme in lignin biosynthesis. The invention further provides a method for altering the lignin composition in plants by transforming plants with the F5H gene wherein the gene is expressed and causes an increased conversion of ferulic acid to sinapic acid thereby increasing the svringyl content of the lignin polymer.

The effect in plants of lignin compositions containing higher syringyl monomer content is that the lignin is more susceptible to chemical delignification.

This is of particular use in the paper and pulp industries where vast amounts of energy and time are consumed in the delignification process. Woody plants transformed with an active F5H gene would offer a significant advantage in the delignification process over conventional paper feedstocks. Similarly, modification of the lignin composition in grasses by the insertion and expression of a heterologus F5H gene offers a unique method for increasing the digestibility of livestock feed. Maximizing the digestibility of grasses in this manner offers great potential economic benefit to the farm and agricultural industries.

Plants to which the Invention may be Applied The invention provides a gene and a chimeric gene construct useful for the transformation of plant tissue for the alteration of lignin monomer composition.

Plants suitable in the present invention comprise plants that naturally lack syringyl lignin or those that accumulate lignin with a high guaiacyl:syringyl ratio. Plants suitable in the present invention also comprise plants whose lignin could be modified using antisense transformation constructs that reduce the syringyl content of the transgenic plants' lignin if such an alteration were desirable.

Suitable plants may include but are not limited to alfalfa (Medicago sp.), rice (Oryza maize (Zea mays), oil seed rape(Brassica forage grasses, and also tree crops such as eucalyptus (Eucalyptus pine (Pinus spruce (Picea sp.) and poplar (Populus as well as Arabidopsis sp. and tobacco •(Nicotiana sp).

20 Definitions As used herein the following terms may be used for interpretation of the claims and specification.

The term "FAH1" refers to the locus or chromosomal location at which the gene is encoded. The term "FAHI" refers to the wild type allele of the gene encoding the F5H gene. The term "fahi" refers to any mutant version of that gene that leads to an altered level of enzyme activity, syringyl lignin content or sinapate ester content that can be measured by thin layer chromatography, high performance liquid chromatography, or by in vivo fluorescence.

"Gene" refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding non-coding) and following noncoding) the coding region. "Native" gene refers to the gene as found in nature with its own regulatory sequences.

A "chimeric gene" refers to a gene comprising heterogeneous regulatory and coding sequences.

An "endogenous gene" refers to the native gene normally found in its natural location in the genome.

A "foreign gene" or "transgene" refers to a gene not normally found in the host organism but one that is introduced by gene transfer.

I' a The term "promoter" refers to a DNA sequence in a gene, usually upstream to its coding sequence, which controls the expression of the coding sequence by providing the recognition site for RNA polymerase and other factors required for proper transcription. A promoter may also contain DNA sequences that are involved in the binding of protein factors which control the effectiveness of transcription initiation in response to physiological or developmental conditions.

The term "operably linked" refers to nucleic acid sequences on a single nucleic acid molecule which are associated so that the function of one is affected by the other.

As used herein, suitable "regulatory sequences" refer to nucleotide sequences located upstream within, and/or downstream of a coding sequence, which control the transcription and/or expression of the coding sequences in conjunction with the protein biosynthetic apparatus of the cell. These regulatory sequences include promoters, translation leader sequences, transcription termination sequences, and polyadenylation sequences.

The term "T-DNA" refers to the DNA that is transferred into the plant genome from a T-DNA plasmid carried by a strain ofAgrobacterium tumefaciens that is used to infect plants for the purposes of plant transformation.

The term "T-DNA plasmid" refers to a plasmid carried by Agrobacteriurn tumefaciens that carries an origin of replication, selectable markers such as antibiotic resistance, and DNA sequences referred to as right and left borders that are required for plant transformation. The DNA sequence that is transferred during this process is that which is located between the right and left T-DNA border sequences present on a T-DNA plasmid. The DNA between these borders can be manipulated in such a way that any desired sequence can be inserted into the plant genome.

The term "ferulate-5-hydroxylase" or "F5H" will refer to an enzyme in the plant phenylpropanoid biosynthetic pathway which catalyzes the conversion of ferulate to 5-hydroxyferulate and permits the production ofsinapic acid and its 30 subsequent metabolites, including sinapoylmalate and syringyl lignin.

The terms "encoding" and "coding" refer to the process by which a gene, through the mechanisms of transcription and translation, provides the information to a cell from which a series of amino acids can be assembled into a specific amino acid sequence to produce an active enzyme. It is understood that the process of encoding a specific amino acid sequence includes DNA sequences that may involve base changes that do not cause a change in the encoded amino acid, or which involve base changes which may alter one or more amino acids, but do not affect the functional properties of the protein encoded by the DNA sequence. It is i; A, therefore understood that the invention encompasses more than the specific exemplary sequences. Modifications to the sequence, such as deletions, insertions, or substitutions in the sequence which produce silent changes that do not substantially affect the functional properties of the resulting protein molecule are also contemplated. For example, alterations in the gene sequence which reflect the degeneracy of the genetic code, or which result in the production of a chemically equivalent amino acid at a given site, are contemplated. Thus, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a biologically equivalent product.

Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the protein molecule would also not be expected to alter the activity of the protein. In some cases, it may in fact be desirable to make mutants of the sequence in order to study the effect of alteration on the biological activity of the protein. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity in the encoded products.

20 Moreover, the skilled artisan recognizes that sequences encompassed by this S. invention are also defined by their ability to hybridize, under stringent conditions (2X SSC, 0.1% SDS, 65 with the sequences exemplified herein.

The term "expression", as used herein, refers to the production of the protein product encoded by a gene. "Overexpression" refers to the production of a gene product in transgenic organisms that exceeds levels of production in normal or non-transformed organisms.

"Transformation" refers to the transfer of a foreign gene into the genome of a host organism and its genetically stable inheritance. Examples of methods of plant transformation include Agrobacterium-mediated transformation and particleaccelerated or "gene gun" transformation technology as described in U.S. 5,204,253.

The term "plasmid rescue" will refer to a technique for circularizing restriction enzyme-digested plant genomic DNA that carries T-DNA fragments bearing a bacterial origin of replication and antibiotic resistance (encoded by the -lactamase gene ofE. coli) such that this circularized fragment can be propagated as a plasmid in a bacterial host cell such as E. coli.

The term "lignin monomer composition" refers to the relative ratios of guaiacyl monomer and syringyl monomer found in lignified plant tissue.

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The Phenvlpropanoid Biosynthetic Pathway The lignin biosynthetic pathway is well researched and the principal pathways are illustrated in Figure 1. Lignin biosynthesis is initiated by the conversion of phenylalanine into cinnamate through the action of phenylalanine ammonia lyase (PAL). The second enzyme of the pathway is cinnamate-4hydroxylase (C4H), a cytochrome P450-dependent monooxygenase (P450) which is responsible for the conversion of cinnamate to p-coumarate. The second hydroxylation of the pathway is catalyzed by a relatively ill-characterized enzyme, p-coumarate-3-hydroxylase (C3H), whose product is caffeic acid. Caffeic acid is subsequently O-methylated by OMT to form ferulic acid, a direct precursor of lignin. The last hydroxylation reaction of the general phenylpropanoid pathway is catalyzed by F5H. The 5-hydroxyferulate produced by F5H is then O-methylated by OMT, the same enzyme that carries out the O-methylation of caffeic acid. This dual specificity of OMT has been confirmed by the cloning of the OMT gene, and expression of the protein in E. coli (Bugos et al., (1991) supra; Gowri et al., (1991) supra).

The committed steps of lignin biosynthesis are catalyzed by 4CL, (hydroxy)cinnamoyl CoA reductase (CCR) and CAD, which ultimately generate coniferyl alcohol from ferulic acid and sinapoyl alcohol from sinapic acid.

Coniferyl alcohol and sinapoyl alcohol are polymerized by extracellular oxidases to yield guaiacyl lignin and syringyl lignin respectively, although syringyl lignin is more accurately described as a co-polymer of both monomers.

Although ferulic acid, sinapic acid, and in some cases p-coumaric acid are channeled into lignin biosynthesis, in some plants these compounds are precursors for other secondary metabolites. In Arabidopsis, sinapic acid serves as a precursor for lignin biosynthesis but it is also channeled into the synthesis of soluble sinapic acid esters. In this pathway, sinapic acid is converted to sinapoylglucose which serves as an intermediate in the biosynthesis of sinapoylmalate (Figure Sinapic acid and its esters are fluorescent and may be use as a marker of plants deficient in 30 those enzymes needed to produce sinapic acid (Chapple et al., supra).

Identification of the FAHI Locus and fahl Alleles A series of mutants of Arabidopsis that fail to accumulate sinapoylmalate have been identified and have been collectively termedfahl mutants. The fluorescent nature of sinapoylmalate permits the facile identification of sinapic acid esters by thin layer chromatography (TLC) followed by observation under ultraviolet (UV) light). The fluorescence of sinapoylmalate can also be visualized in vivo because sinapoylmalate is accumulated in the adaxial leaf epidermis. Wild type Arabidopsis exhibits a pale blue fluorescence under LUV whilefahl mutants n I appear dark red because of the lack of the blue fluorescence of sinapoylmalate and the fluorescence of chlorophyll in the subtending mesophyll (Chapple et al., supra).

A TLC-based mutant screen of 4.200 ethyl methanesulfonate-mutagenized Arabidopsis plants identified a number of independent mutant lines that accumulated significantly lower levels ofsinapoylmalate. The mutations in these lines were identified asfahl-1 throughfahl-5. The in vivo UV-fluorescence visual screen was used to identify more mutant lines carrying thefahl mutation. Two of these mutants (fahl-6 andfahl-7) were selected from EMS-mutagenized populations. One mutant line (fahl-8) was selected from among a mutant population generated by fast-neutron bombardment (Nilan, R. A. Nucl Sci. Abstr., 28(3), 5940 (1973); Kozer et al., Genet. Pol., 26(3),367, (1985)). A final mutant line, (fahl-9) was identified using the same technique from a T-DNA tagged population of plants. Before further analysis, each mutant line was backcrossed at least twice to the wild type and homozygous lines were established.

To determine whether the newly isolated mutant lines were defective at the same locus, that is, within the gene encoding F5H, genetic complementation experiments were performed. In these tests, each mutant line was crossed to fahl-2 which is known to be defective in F5H. In each case, the newly isolated mutant line was used as the female parent and was fertilized with pollen from a 20 fahl-2 homozygous mutant. A reciprocal cross was also performed usingfahl-2 as the female parent, and the new mutant line as the pollen donor. The seeds from these crosses were collected several weeks later, and were planted for subsequent analysis. The progeny were analyzed for sinapoylmalate production by TLC, high pressure liquid chromatography and by observation under UV light. From these crosses, all of the Fl progeny examined were sinapoylmalate-deficient, indicating that all of the mutations identified were allelic.

Thefahl-9 line was selected for further study because of the presence of the T-DNA insertion within the F5H gene. The T-DNA insertion within the FAH1 locus facilitated the cloning of the flanking Arabidopsis DNA which could then be used to retrieve the wild type F5H gene from cDNA and genomic libraries (Meyer et al., Proc. Natl. Acad Sci. USA, 93, 6869 (1996)).

Cloning of the FAH1 Locus A fragment of DNA from the FAHI locus was isolated from the T-DNA taggedfahl-9 mutant using the technique ofplasmid rescue (Meyer et al., supra) The technique of plasmid rescue is common and well known in the art and may be used to isolate specific alleles from T-DNA transformed plants (Behringer, et al., Plant Mol. Biol. Rep., 0, 190,(1992)). Briefly, the vector used to generate the T-DNA tagged population of Arabidopsis carries sequences required for I autonomous replication of DNA in bacteria and sequences that confer antibiotic resistance. Once this DNA is integrated into the plant genome, specific restriction endonuclease digests can be employed to generate fragments that can be circularized, ligated, and transformed into E. coli. Circularized DNA from the T-DNA will generate functional plasmids that confer antibiotic resistance to their bacterial hosts such that they can be identified by growth on selective media.

Those plasmids that are generated from the sequences including the right and left borders will also carry with them the plant genomic sequences flanking the T-DNA insertion. Plasmids generated from either of the T-DNA borders that carry flanking DNA sequences can be identified by analyzing the products of diagnostic restriction enzyme digests on agarose gels. The plasmids with flanking sequences can then serve as a starting point for cloning plant sequences that share homology to the DNA at the point of T-DNA insertion (Behringer, et al., supra).

Plasmid rescue was conducted using EcoRI-digested DNA prepared from homozygousfahl-9 plants. EcoRI-digested genomic DNA was ligated and then electroporated into competent DH5ac E coli. DNA from rescued plasmids was further digested with both EcoRI and Sail and the digests were analyzed by gel "electrophoresis to identify plasmids that contained flanking Arabidopsis DNA. A SacII-EcoRI fragment from this rescued plasmid was used to identify an F5H clone S 20 from an Arabidopsis cDNA library (Newman, T. et al., Plant. PhysioL 106, 1241, (1994)).

DNA Sequencing of the F5H cDNA and genomic clones Sequence analysis of the F5H cDNA and genomic clones was performed on plasmid DNA manually using a United States Biochemical Sequenase Kit v. 2.0, on S 25 a DuPont Genesis® 2000 sequencer or on an Applied Biosystems 373A DNA sequencer, using standard vector-based sequencing oligonucleotides or customsynthesized oligonucleotides as appropriate. The sequence of the Arabidopsis thaliana FSH cDNA is given in SEQ ID NO.:1 and the sequence of the Arabidopsis thaliana F5H genomic clone is given in SEQ ID NO.:3.

30 The F5H cDNA contains a 1560 bp open reading frame that encodes a "protein with a molecular weight of 58,728. The putative ATG initiation codon is flanked by an A at -3 and a G at in keeping with the nucleotides commonly found flanking the initiator methionine in plant mRNAs (Lutcke et al., EMBO J. 6, 43, (1987)). Immediately following the inferred initiator methionine is a 17 amino acid sequence containing nine hydroxy amino acids (Figure The subsequent fifteen amino acid sequence is rich in hydrophobic amino acids; eleven hydrophobic residues comprised of phenylalanine. isoleucine, leucine and valine residues. This hydrophobic stretch is immediately followed by an Arg-Arg-Arg-Arg putative stop transfer sequence. F5H also shares significant sequence identity with other P450s.

Most notable is the stretch between Pro-450 and Gly-460. This region contains eight residues that comprise the heme-binding domain and are highly conserved among most P450s, one exception being allene oxide synthase from Linum usitatissimum (Song et al., Proc. Natl. Acad Sci. USA 90. 8519, (1993)). The Pro-450 to Gly-460 region contains Cys-458 in F5H, which by analogy is most likely the heme binding ligand in this enzyme.

Transformation of fahl-2 Arabidopsis and Restoration of Sinapovlmalate Accumulation The identity of the F5H gene was confirmed by complementation of the fahl-2 mutant with a genomic clone and a construct where the F5H genomic coding sequence was expressed under the control of the cauliflower mosaic virus promoter. Briefly, the F5H cDNA was used as a probe to screen a transformation competent library (Meyer et al., (1994) Science, 264, 1452-1455) for genomic clones. Using this method, a cosmid clone (pBIC20-F5H) was isolated that carried a 17kb genomic insert containing the inferred start and stop codons of the F5H gene (Figure The portion of this cosmid carrying the open reading frame was excised from the cosmid and subcloned into a vector in which it was operably linked to the cauliflower mosaic virus 35S promoter 20 (pGA482-35S-F5H) (Figure Both the original cosmid and this derivative plasmid construct were electroporated into Agrobacterium tumefaciens and were used to transformfahl-2 mutants. Success of the transformations was evidenced by TLC assays demonstrating sinapoylmalate accumulation in leaf tissues of the fahl-2 transformants carrying the T-DNA from the pBIC20-F5H cosmid or the pGA482-35S-F5H plasmid (Figure These data clearly indicated that the gene encoding F5H had been identified.

Modification of Lienin Composition in Plants Transformed With F5H Under the Control of the Cauliflower Mosaic Virus 35S Promoter Arabidopsis plants homozygous for thefahl-2 allele were transformed with Agrobacterium carrying the pGA482-35S-F5H plasmid which contains the "chimeric F5H gene under the control of the constitutive cauliflower mosaic virus 35S promoter (Odell. et al., Nature 313, 810-812, (1985)). Independent homozygous transformants carrying the F5H transgene at a single genetic locus were identified by selection on kanamycin-containing growth media, grown up in soil and plant tissue was analyzed for lignin monomer composition. Nitrobenzene oxidation analysis of the lignin in wild type, fahl-2, and transformants carrying the T-DNA from the pGA482-35S-F5H construct revealed that F5H overexpression as measured by northern blot analysis (Figure 4) led to a significant increase in syringyl content of the transgenic lignin (Figure The lignin of the plants demonstrated a syringyl content as high as 29 mol% as opposed to the syringyl content of the wild type lignin which was 18 mol% (Table 1) (Example These data clearly demonstrate that overexpression of the FSH gene is useful for the alteration of lignin composition in transgenic plants.

TABLE I Impact of 35S Promoter-Driven F5H Expression on Lienin Monomer Corminsition in Arabidoni Total G units 3 Line (wnol 2-1 d.w.) wild type 3.33 0.32 fahl-2 5.4-4 +10.45 88 6.63 +10.75 172 4.21+10.36 170 4.08 +-0.33 122 3.74+/0.20 108 5.40 +-0.48 107 5.74 +-0.60 180 3.85+/0.31 117 3.21 +10.30 128 3.46+10.22 as=m of vanillin vanillic acid Total S units 0 0.75 0.09 nad.

0.35 +-0.04 0.67 +-0.07 0.97 +-0.06 0.93 +10.05 1.59 18 1.96 10.31 1.34 +-0.11 1. 18 0. 13 1.39 0.17 Total G+S units (I.±mol g-1 d w.) mol% S 4.09 +-0.41 5.44 1-0.45 6.99 +-0.79 4.88 +-0.42 5.05 +-0.37 4.66 +-0.22 6.98 +-0.65 7.70 +-0.89 5.19 +-0.40 4.39 +-0.43 5.05 +-0.37 18.4 +-0.91 5.06 17 13.7 +-0.55 19.2 0.56 19.9 0.86 22.7 0.82 25.3 1.23 25.8 0.78 28.8 +-0.92 27.5 1-1.80 *too *000 000.

0O 0 bsumi of syringaldehyde syringic acid In a simila fashion, T I tobacco (Nicofiana tabacum) F5H transformants were generated, grown up and analyzed for ignin monomer composition.

Nitrobenzene oxidation analysis demonstrated that the syringyl monomer content of the leaf midribs was increased from 14 mol% in the wild type to 40 mol% in the transgenic line that most highly expressed the F5H trantsgene (Table 2).

TABLE2 Impact of 35 S Promoter-Driven F5H Expression on Lignin Monomer Composition in Tobacco Leaf Midrib Xylem Total G units 3 Total S titb Total G+S units Line mol g-1 (pgrxol a- (4mol e- mol S wild type 1.40 -26 0.23 +10.04 1.63 -10.30 14.3 1.09 0.86 /0.16 0.24 -0.03 1.11 +-0.20 22.4 +-1.53 27 1.13+/0.11 0.52 I0.05 1.65+10.16 31.3 /0.50 48 1.28 I-0.32 0. 71 0. 19 1.99 1-0.43 35.7 *I-6.06 33 0.65 0.17 0.43 1.09 -10.27 40.0 +-1.86 asuin of vanillin vamullic acid bsujm of syringaidehyde syringic acid Construction of Chimeric Genes for the Expression of F5H in Plants.

The expression of foreign genes in plants is well-established (De Blaere et al. (1987) Mleth. Enzvmol. 143:277-291) and this invention provides for a method to apply this technology to the introduction of a chimeric gene for the overexpression of the F5H gene in plants for the manipulation of lignin monomer composition. The expression of the F5H mRNAs at an appropriate level may require the use of different chimeric genes utilizing different promoters. A preferred class of heterologous hosts for the expression of the coding sequence of the F5H gene are eukaryotic hosts, particularly the cells of higher plants.

Particularly preferred among the higher plants and the seeds derived from them are alfalfa (Medicago rice (Oryza maize (Zea mays), oil seed rape (Brassica forage grasses, and also tree crops such as eucalyptus (Eucalyptus pine (Pinus spruce (Picea sp.) and poplar (Populus as well as Arabidopsis sp.

and tobacco (Nicotiana Expression in plants will use regulatory sequences functional in such plants.

The origin of the promoter chosen to drive the expression of the coding sequence is not critical as long as it has sufficient transcriptional activity to accomplish the invention by expressing translatable mRNA for the F5H gene in the desired host tissue. Preferred promoters will effectively target F5H expression to those tissues that undergo lignification. These promoters may include, but are not limited to promoters of genes encoding enzymes of the phenylpropanoid pathway such as the PAL promoter (Ohi et al., Plant Cell, 2, 837, (1990) and the 4CL promoter(Hauffed et al., Plant Cell, 3, 435, (1991).

25 Depending upon the application, it may be desirable to select promoters that are specific for expression in one or more organs of the plant. Examples include the light-inducible promoters of the small subunit of ribulose carboxylase, if the expression is desired in photosynthetic organs, or promoters active specifically in roots.

Expression ofFSH Chimeric Genes in Plants Various methods of introducing a DNA sequence of transforming) into eukaryotic cells of higher plants are available to those skilled in the art (see EPO publications 0 295 959 A2 and 0 138 341 Al). Such methods include those based on transformation vectors based on the Ti and Ri plasmids of Agrobacterium spp. It is particularly preferred to use the binary type of these vectors. Ti-derived vectors transform a wide variety of higher plants, including monocotyledonous and S* dicotyledonous plants, such as soybean, cotton, tobacco, Arabidopsis and rape (Pacciotti et al., Bio/Technology 3, 241, (1985); Byre et al., Plant Cell, Tissue and Organ Culture 8, 3, (1987); Sukhapinda et al., Plant Mol. Biol. 8, 209, (1987). Lorz et al.. Mol. Gen. Genet. 199. 178, (1985), Potrvkus Wol. Gen. Genet 199, 183, (1985)).

For introduction into plants the chimeric genes of the invention can be inserted into binary vectors as described in Example Other transformation methods are available to those skilled in the art, such as direct uptake of foreign DNA constructs [see EPO publication 0 295 959 A2], techniques of electroporation [see Fromm et al. (1986) Nature (London) 319:791] or high-velocity ballistic bombardment with metal particles coated with the nucleic acid constructs (see Kline et al., Nature (London) 327:70 (1987), and see U.S.

Patent No. 4,945,050). Once transformed, the cells can be regenerated by those skilled in the art.

The following Examples are meant to illustrate key embodiments of the invention but should not be construed to be limiting in any way.

EXAMPLES

GENERAL METHODS Restriction enzyme digestions, phosphoryiations, ligations and transformations were done as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press.

All reagents and materials used for the growth and maintenance of bacterial cells were obtained from Aldrich Chemicals (Milwaukee, WI), DIFCO Laboratories (Detroit, MI), GIBCO/BRL (Gaithersburg, MD), or Sigma Chemical Company (St. Louis, MO) unless otherwise specified.

The meaning of abbreviations is as follows: means hour(s), "min" means minute(s), "sec" means second(s), means day(s), "pL" means 25 microliter(s), "mL" means milliliters, means liters, means grams, "mg" means milligrams, means microgram(s), "nm" means nanometer(s), "m" means meter(s), means Einstein(s).

Plant material Arabidopsis thaliana was grown under a 16 h light/8 h dark photoperiod at 100 mE m- 2 sl at 24 °C cultivated in Metromix 2000 potting mixture (Scotts, Marysville OH). Mutant linesfahl-I throughfahl-5 were identified by TLC as described below. Using their red fluorescence under UV light as a marker, mutant linesfahl-6,fahl-7, andfahI-8 were selected from ethylmethane sulfonate (fahl-6, S fahl-7) or fast-neutron (fahl-8) mutagenized populations ofLandsberg erecta M2 35 seed. The T-DNA tagged line 3590 (fahl-9) was similarly identified in the DuPont T-DNA tagged population (Feldmann, Malmberg, Dean, (1994) Muragenesis in Arabidopsis in Arabidopsis, M. Meyerowitz and C. R.

Somerville, eds.) Cold Spring Harbor Press). All lines were backcrossed to wild type at least twice prior to experimentai use to remove unliniked background mutations.

Secnda etabolite Anass Leaf extracts were prepared from 100 gsmls Freshd lnef tissefoe suspended in I mrL of 50%/ methanol. Samples were vortxdbify hnfoe at -70 0 C. Samples were thawed, vortexed, and cenrtifuged at 12,000 xg for min. Sinapoylmfalate content was qualitatively determined following silica gel TLC, in a mobile phase of n-butanol/ethanoVwater SinapiC acid and its esters were visualized under long wave bTV light (365 r) by their characteristic fluorescence.

Southern lsisi For Southern analysis, DNA was extracted from leaf material (Rogers, et al., (1985) Plant. Mol. Biol 5, 69), digested with restrictionl endoflelases and tran-sferred to Hybnd N+ membrane (AmeYshlID' Cleveland Ohio) by standard protocols. CDNA probes were radiolabelled with 3 Padhbiieotetre membrane in Denhardt's hybridization buffer (900 mm sodiumi chloride, 6 mM disodjuin EDTA, 60 mM sodium phosphate pHl 7-4, 0.5% SD 0.01% denlatured herring sperm DNA and 0. 1% each poiyvinylpyrrolidone, bovine serum albumin, and Ficoll 400) containing 50% forinanide at 42 0 C. To remove unbound probe, membranes were washed twice at room temperaturle and twice at 65 0 C in 2x 55SPE (300 MtM sodium chloride, 2 muM disodluml EDTA, 20mrM sodium phosphate, pHi 7.4) containing 0. 1% SDS, and exposed- to 6lin.

Northern Analyi was first extracted from leaf material according to the following *:25 protocol.

For extraction of RNA, Coveys extraction buffer was prepared by dissolving 1% TIPS (triisopropyl nphthiefe sulfonate, sodium salt), 6% pAS (p-aninosalicylate, sodium salt) in 50 mM Tris pH 8.4 containing v/v Kirby's phenol. Kirby's phenol was prepared by neutralizinlg liquified phenol *30 containing 0. 1% (wlv) 8-hydroxyquinofine with 0. 1 M TrisHCI pHI 8.8. For each RNA preparation,~ a I g samples of plant tissue was ground in liquid nitrogen and extracted in 5 niL Covey's extraction buffer contafiing l0 p1. P-mercaptoethanol.

The sample was extracted with 5 mL of a 1: 1 mixture of Kirbys phenol and chloroformi, vortexed. and centrifuged for 20 min at 7,000 xg. The supernatant was removed and the nucleic acids were preciptae Iit 5 00 o3 Mg foiu acetate and 5 mnL isopropaflol and collected by ceattr0,000tignfor rMi. The pellet was redissolved in 500 p1.. water, and the RQNA was precipitated on ice with 250 pL 8 'M LiCi, and collected by cenltrifugationl at I 10,000 xg for 10 min. The pellet was resuspended in 200 uL water and extracted with an equal volume ofchloroform:isoamyl alcohol 1: with vortexing. After centrifugaion for 2 min at 10,000 xg, the upper aqueous phase was removed, and the nucleic acids were precipitated at -20 °C by the addition of 20 pL 3 M sodium acetate and 200 pL isopropanol. The pellet was washed with I mL cold ethanol, dried, and resuspended in 100 uL water. RNA content was assayed spectrophotometrically at 260 nm. Samples containing 1 to 10 pg of RNA'were subjected to denaturing gel electrophoresis as described elsewhere (Sambrook et al., supra).

Extracted RNA was transferred to Hybond N membrane (Amersham, Cleveland Ohio), and probed with radiolabelled probes prepared from cDNA clones. Blots were hybridized overnight, washed twice at room temperature and once at 65 *C in 3x SSC (450 mM sodium chloride, 45 mM sodium citrate, pH 7.0) containing 0.1% SDS, and exposed to film.

Identification of cDNA and Genomic Clones cDNA and genomic clones for F5H were identified by standard techpiques using a 2.3 kb SaclIEcoRI fragment from the rescued plasmid (pCC1) (Example 2) as a probe. The cDNA clone pCC30 was identified in the LPRL2 library (Newman et al., (1994) supra) kindly provided by Dr. Thomas Newman (DOE Plant Research Laboratory, Michigan State University, East Lansing, MI).

A genomic cosmid library ofArabidopsis thaliana (ecotype Landsberg erecta) generated in the binary cosmid vector pBIC20 (Example 3) (Meyer et al., Science 264, 1452, (1994)) was screened with the radiolabelled cDNA insert derived from pCC30. Genomic inserts in the pBIC20 T-DNA are flanked by the neomycin 25 phosphotransferase gene for kanamycin selection adjacent to the T-DNA right border sequence, and the P-glucuronidase gene for histochemical selection adjacent to the left border. Positive clones were characterized by restriction digestion and Southern analysis in comparison to Arabidopsis genomic DNA.

Plant transformation 30 Transformation of Arabidopsis thaliana was performed by vacuum infiltration (Bent et al., Science 265, 1856, (1994)) with minor modifications.

Briefly, 500 mL cultures of transformed Agrobacterium harboring the pBIC20-F5H cosmid or the pGA482-35S-FSH construct were grown to stationary phase in Luria broth containing 10 mg L- 1 rifampicin and 50 mg L- 1 kanamycin.

Cells were harvested by centrifugation and resuspended in 1 L infiltration media containing 2.2 g MS salts (Murashige and Skoog, Physiol. Plant. 15, 473, (1962)), Gamborg's B5 vitamins (Gamborg et al., Exp. CellRes. 50, 151, (1968)), 0.5 g MES. 50 g sucrose, 44 rM benzylaminopurine, and 200 uL Silwet L-77 (OSI Specialties) at pH 5.7 Bolting Arabidopsis plants (To generation) that were 5 to cm tall were inverted into the bacterial suspension and exposed to a vacuum (>500 mm ofHg) for three to five min. Infiltrated plants were returned to standard growth conditions for seed production. Transformed seedlings

(T

1 were identified by selection on MS medium containing 50 mg L-1 kanamycin and 200 mg L- 1 timentin (SmithKline Beecham) and were transferred to soil.

Transformation of tobacco was accomplished using the leaf disk method of Horsch et al. (Science 227, 1229, (1985)).

Nitrobenzene oxidation For the determination of lignin monomer composition, stem tissue was ground to a powder in liquid nitrogen and extracted with 20 mL of 0.1 M sodium phosphate buffer, pH 7.2 at 37 *C for 30 min followed by three extractions with ethanol at 80 The tissue was then extracted once with acetone and completely dried. Tissue was saponified by treatment with 1.0 M NaOH at 37 *C for 24 hours, washed three times with water, once with 80% ethanol, once with acetone, and dried. Nitrobenzene oxidation of stem tissue samples was performed with a protocol modified from liyama et al. Sci. FoodAgric. 51, 481-491.

(1990)). Samples of lignocellulosic material (5 mg each) were mixed with 500 .L of 2 M NaOH and 25 .iL of nitrobenzene. This mixture was incubated in a sealed 20 glass tube at 160 *C for 3 h. The reaction products were cooled to room temperature and 5 pL of a 20 mg mL-1 solution of 3-ethoxy-4-hydroxybenzaldehyde in pyridine was added as an internal standard before the mixture was extracted twice with 1 mL ofdichloromethane. The aqueous phase was acidified with HCI (pH 2) and extracted twice with 900 iL of ether. The combined ether phases were dried with anhydrous sodium sulfate and the ether was evaporated in a stream of nitrogen. The dried residue was resuspended in 50 pL of pyridine, 10 uL ofBSA (N,O-bis-(trimethylsilyl)-trifluoracetamide) was added and 1 IL aliquots of the silylated products were analyzed using a Hewlet-Packard 5890 Series II gas chromatograph equipped with Supelco SPB I column (30 m x 30 0.75 mm). Lignin monomer composition was calculated from the integrated areas of the peaks representing the trimethylsilylated derivatives of vanillin, syringaldehyde, vanillic acid and syringic acid. Total nitrobenzene oxidationsusceptible guaiacyl units (vanillin and vanillic acid) and syringyl units (syringaldehyde and syringic acid) were calculated following correction for recovery efficiencies of each of the products during the extraction procedure relative to the internal standard.

I rl EXAMPLE I IDENTIFICATION OF THE T-DNA TAGGED ALLELE OFFAHI A putatively T-DNA taggedfahl mutant was identified in a collection of T-DNA tagged lines (Feldmann et al., Mol. Gen. Genet. 208, 1, (1987)) (Dr. Tim Caspar, Dupont, Wilmington, DE) by screening adult plants under long wave UV light. A red fluorescent line (line 3590) was selected, and its progeny were assayed for sinapoylmalate content by TLC. The analyses indicated that line 3590 did not accumulate sinapoylmalate. Reciprocal crosses of line 3590 to afahl-2 homozygote, followed by analysis of the F generation for sinapoylmalate content demonstrated that line 3590 was a new allele offahl, and it was designatedfahl-9.

Preliminary experiments indicated co-segregation of the kanamycinresistant phenotype of the T-DNA tagged mutant with thefahl phenotype. Selfed seed from 7 kanamycin-resistant (fahl-9 x FAH1] Fl plants segregated 1:3 for kanamycin resistance (kanr i tv e knarsman) and 3:1 for sinapoylmalate deficiency (FAHl:fahl). From these lines, fahl plapts gave rise to only kan r esu r ant fahl progeny. To determine the genetic distance between the T-DNA insertion and the FAH1 locus, multiple test crosses were performed between a [fahl-9 x FAHI] Fl and afahl-2 homozygote. The distance between the FAH1 locus and the T-DNA insertion was evaluated by determining the frequency at which 20 FAHl/kar e n O v e progeny were recovered in the test cross Fl. In the absence of crossover events, all kanamycin-resistant Fl progeny would be unable to accumulate sinapoylmalate, and would thus fluoresce red under UV light. In 682 karsistant F progeny examined, no sinapoyimalate proficient plants were identified, indicating a very tight linkage between the T-DNA insertion site and the 25 FAH1 locus.

EXAMPLE 2 PLASMID RESCUE AND cDNA CLONING OF THE fahl GENE S. Plasmid rescue was conducted using EcoRI-digested DNA prepared from homozygousfahl- 9 plants (Behringer et al., (1992), supra). Five tg of EcoRI-digested genomic DNA was incubated with 125 U T4 DNA ligase overnight at 14 *C in a final volume of I mL. The ligation mixture was S. concentrated approximately four fold by two extractions with equal volumes of 2-butanol, and was then ethanol precipitated and electroporated into competent cells as described (Newman et al., (1994), supra).

DNA from rescued plasmids was double digested with EcoRI and Sall.

Plasmids generated from internal T-DNA sequences were identified by the presence of triplet bands at 3.8, 2.4 and 1.2 kb and were discarded. One plasmid (pCCI) giving rise to the expected 3.8 kb band plus a novel 5.6 kb band was I -I

I

identified as putative external right border plasmid. Using a SaclLEcoRI fragment of pCC I that appeared to represent Arabidopsis DNA, putative cDNA clones for F5H were identified. The putative FSH clone carried a 1.9 kb Sall-NotI insert, the sequence of which was determined. Blastx analysis (Altschul et al., J Mol. Biol. 215, 403, (1990)) indicated that this cDNA encodes a cytochrome P450-dependent monooxygenase, consistent with earlier reports that thefahl mutant is defective in F5H (Chapple et al., supra) and (ii) F5H is a cytochrome P450-dependent monooxygenase (Grand, supra).

Southern and Northern Blot analysis To determine whether the putative F5H cDNA actually represented the gene that was disrupted in the T-DNA tagged line Southern and northern analysis was used to characterize the availablefahl mutants using the putative F5H cDNA.

Figure 6 shows a Southern blot comparing hybridization of the F5H cDNA to EcoRI-digested genomic DNA isolated from wild type (ecotypes Columbia (Col), Landsberg erecta (LER), and Wassilewskija and the ninefahl alleles including the T-DNA taggedfahl-9 allele. WS is. the ecotype from which the T-DNA tagged line was generated.

These data indicated the presence of a restriction fragment length polymorphism between the tagged line and the wild type. These data also indicates a restriction fragment length polymorphism in thefahl-8 allele which was generated with fast neutrons, a technique reported to cause deletion mutations.

As shown in Figure 6 the genomic DNA ofthefahl-8 andfahl-9 (the T-DNA tagged line) alleles is disrupted in the region corresponding to the putative S. F5H cDNA. These data also indicate that F5H is encoded by a single gene in 25 Arabidopsis as expected considering that the mutation in thefahl mutant Ssegregates as a single Mendelian gene. These data provide the first indication that the putative F5H cDNA corresponds to the gene that is disrupted in thefahl mutants.

Plant material homozygous for nine independently-derivedfahl alleles was surveyed for the abundance of transcript corresponding to the putative F5H cDNA using Northern blot analysis. The data is shown in Figure 7.

As can be seen from the data, the putative F5H mRNA was represented at similar levels in leaf tissue of Columbia, Landsberg erecta and Wassilewskija ecotypes. and in the EMS-inducedfahl-1, fahl-4, andfahl-5, as well as the fast 35 neutron-inducedfahl- Transcript abundance was substantially reduced in leaves from plants homozygous for thefahl-2,fahl-3 andfahl-6, all of which were EMS-induced, the fast neutron-induced mutantfahl-S and in the tagged linefahl-9.

The mRNA infahl-8 mutant also appears to be truncated. These data provided strong evidence that the cDNA clone that had been identified is encoded by the FAHI locus.

EXAMPLE 3 DEMONSTRATION OF THE IDENTITY OF THE F5H cDNA BY TRANSFORMATION OF fahl MUTANT PLANTS WITH WILDTYPE AND RESTORATION OF SINAPOYLMALATE ACCUMULATION In order to demonstrate the identity of the F5H gene at the functioal level, the transformation-competent pBIC20 cosmid library (Meyer et al., supra) was screened for corresponding genomic clones using the full length F5H cDNA as a probe. A clone (pBIC20-FSH) carrying a genomic insert of 17 kb that contains 2.2 kb of sequence upstream of the putative F5H start codon and 12.5 kb of sequence downstream of the stop codon of the F5H gene (Figure 2) was transformed into thefahl-2 mutant by vacuum infiltration. Thirty independent infiltration experiments were performed, and 167 kanamycin-resistant seedlings, representing at least 3 transformants from each infiltration, were transferred to soil and were analyzed with respect to sinapic acid-derived secondary metabolites. Of these plants, 164 accumulated sinapoylmalate in their leaf tissue as determined by TLC (Figure These complementation data indicate that the gene defective in thefall mutant is present on the binary cosmid To delimit the region of DNA on the pBIC20-FSH cosmid responsible for complementation of the mutant phenotype, a 2.7 kB fragment of the FSH genomic sequence was fused downstream of the cauliflower mosaic virus 35S promoter in the binary plasmid pGA482 and this construct (pGA482-35S-F5H) (Figure 2) was transformed into thefahl-2 mutant. The presence of sinapoylmalate in 109 out of 110 transgenic lines analyzed by TLC or by in vivo fluorescence under UV light indicated that thefahl mutant phenotype had been complemented (Figure 3).

These data provide conclusive evidence that the F5H cDNA has been identified.

EXAMPLE 4 DNA SEQUENCING OF THE F5H cDNA AND GENOMIC CLONES 30 The FSH cDNA and a 5156 bp HindIII-Xhol fragment of the pBIC20-F5H genomic clone were both fully sequenced on both strands and the sequence of the F5H protein (SEQ ID NO.:2) was inferred from the cDNA sequence (Figure 8).

The sequence ofthe.Arabidopsis thaliana F5H cDNA is given in SEQ ID NO.: 1.

The sequence of the Arabidopsis thaliana F5H genomic clone is given in SEQ ID NO.:3.

EXAMPLE MODIFICATION OF LIGNTN MONOMER COMPOSITION IN TRANSGENIC PLANTS OVEREXPRESSING F51H Generation of Transgenic Plants Ectopically Expressing the F5H Gene Using an adaptor-based cloning strategy, regulatory sequences 5' of the translation initiation site of the F5H gene were replaced with the strong constitutive cauliflower mosaic virus 35S promoter (Odell et al., Nature 313, 810-812. (1985)), as shown in Figure 2. The resulting construct carries 2719 bp of the F5H genomic sequence driven by the cauliflower mosaic virus 35S promoter fused 50 bp upstream of the inferred ATG start codon. As a result, the cauliflower mosaic virus 35S promoter drives the expression of the F5H gene by using the transcription start site of the viral promoter and the termination signal present on the F5H genomic sequence. This expression cassette for ectopic expression of was inserted into the T-DNA of the binary vector pGA482 (An, G. (1987), Binary Ti vectors for plant transformation and promoter analysis in: Methods in enzymologv. Wu, R ed. Academic Press, NY 153: 292-305) and introduced into Agrobacterium tumefaciens by electroporation.

Transgenic Arabidopsis plants of the ecotype Columbia that were homozygous for thefahl-2 (Chapple et al., supra) allele were transformed with Agrobacterium cultures harboring the pGA482-35S-F5H construct according to the method of Bent et al. (supra). Transgenic plants of the T2 and T3 generation S. were identified by selection on media containing kanamycin and subsequently S* transferred to soil.

Determination oflignin monomer composition ofArabidopsis stem tissue 25 Total stem tissue was harvested from 4 week old plants that had been grown in soil at 22 *C under a 16 h/8 h light/dark photoperiod. Nitrobenzene oxidation analysis generated mol% syringyl values for 9 different transformant lines (Table 1) ranging from 5.06 0.17 mol% to 28.8 0.92 mol% as opposed to the wildtype control which demonstrated a value of 18.4 0.91 mol%. The 30 fahl-2 mutant background in which the transgenic lines were generated completely lacks syringyl lignin (Table The low expression of the F5H transgene in a genetic background that lacks endogenous F5H message explains how line 88 can have syringyl lignin levels that are lower than wild type.

In addition to Arabidopsis, tobacco plants were transformed in a similar fashion with the F5H gene under control of the cauliflower mosaic virus promoter. T2 and T3 positive transformants were screened and analyzed for lignin modification and the data is given in Table 2. Nitrobenzene oxidation analysis of tobacco leaf midribs generated mol% syringyl values for 4 different transformant lines (Table 2) ranging from 22.4 1.53 mol% to 40.0 1 86 mol% as opposed to the wildtype conltrol which demonstrated a value of 14.3 1.09 mol%.

The data in Tables I and 2 clearly demonstrate that over-e xpressiofl of the FSH gene in transgellc plants results in the modification of lignin monomer composition. The transformed plant is reasonably expected to have syringyl lignin monomer content that is from about 0 mol% to about 95 mol% as measured in whole plant tissue.

*24 SEQUENCE LZSTING GENERAL INFORMATION:

APPLICANT:

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TELEX:

(ii) TITLE OF INVENTION: A METHOD FOR REGULATION OF PLANT LIGNIN COMPOSITION (iii) NUMBER OF SEQUENCES: 3 (iv) COMPUTER READABLE FORM: MEDIUM TYPE: DISKETTE, 3.50 INCH COMPUTER: IBM PC COMPATIBLE OPERATING SYSTEM: WINDOWS 3.1 SOFTWARE: MICROSOFT WORD 2.OC CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:

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K,

2'JFORMATION FOR SEQ :oD NO:7: (i'l SEQUENCE CHFARACTERISTICS: LENGTH: 1838 base paizs TYPE: nucleic aci.d STRANDEDNESS: si ng.1e TOPOLOGY: iinea: (ii) MOLECULE TYPE: DNA (cOMA) (xi) SEQUENCE DESCRIPTION: AAAAAAAACA CTCAATATGG AGTCTTCTAT ATCACAAAC; *0

CACGACGTCT

GCGAAGGCCT

GATGGACCAA

CCATCTCCGC

AGTCCTTCAA

TCTGACTTAC

GAG.AAAAGTG

TCGTGATGAA

CGTCGGGGAG

AGCCTGCGAG

TGGAGCCTTC

AAACAAGCGG

TGAACATATG

CGATATGGTT

AGCGGATCTT

CGTTATGTTT

ATTACGGAGC

TGACAGACGA

AGAAACCCTA

TAGTATcGAc

AGGACGCGAC

ACCGGGCGTAC

TAGATCGTGC C ATTACATTGC TI TGATGTGTTT C

CTTGTC.ATCG

CCATATCCTC

CTCACCCACC

ATGGGATTCC

GTCCAAGACA

GACCGAGCGG

TGTGTCATGA

GTGGACAAAA

CAAATTTTTG

APLGGGACAAG

AACGTAGCGG

CTCGTGAAGG

AAGAAGAAGG

GATGATCTTC

CAAAATTCCA

GGAGGAACGG

CCCGAGGATC

GTTGAAGAAT

kGGATGCACC

;GTTTCTTCA

:CAACCTCTT

:CGGATTTCA

:CGGGTATGC

~TCACGTGGA

;GTCTC.3CGGC

TTGTCTCT.CT

CCGGTCCACG

GTGGTTTAGC

TCCATAa'GTA

GCGTCTTCTC

ACATGGCTTT

AGGTGTTTAG

TGGTCCGGTC

CACTGACCCG

ACGAGTTCAT

ATTTCATACC

CCC GTAATGA

AGAATCAAAA

TTGCTTTTTA

rCAAACTTAC kAACGGTAGC

TAAAACGGGT

CCGACATCGA

-!CCGATCCC

LTCCCAAGAA

;GACTGACCC

kAGGGAGCAA kACTAGGGTT- ATTAC CT GA

TCCTAAAGC

TTTC-ATCTTC

AGGTTGGCCC

CAkATTTAGCI CGCrGTCTCA

GAACCGGCCT

CGCTCACTAC

CCGTAAAAGA

GGTCTCTTGT

CAACATAACT

AAGAATCTTA

ATATTTCGGG

TCTAGACGGA

CGCTGTGGAT

CAGTGAAGAG

CCGTGACAAT

GTCGGCGATA

CCAACAAGAA

GAAGTTGACT

TCTC CT CCT C AzTCTCGTGTG

GGACACGTTT

TTTCGAGTTT

ANTACGCGCTT

TGGGATGAAA

CACGCGGCTT

SEQ ID NO:'

CTAAGCAAAC%

:ATCAGCTTC?

:ATCATAGGCP

AAAAAGTATG

TCACCCGAGG

GCAACTATAG

GGACCGTTTTT

GCTGAGTCAT

AACGTTGGTA

TACCGGGCAG

CAAGAGTTCT

TGGATCGATC

TTTATTGACG

GATGGGGATG

GCCAAATTAG

ATCAAAGCAA

GAGTGGGCCT

CTCGCCGAAG

TATC-TCA.AAT

CACGAAACCG

ATGATCAACG

AGAC CAT CGA

ATACCGTTCG

GACTTAGCCG

C CAAGTGAGC

TTCGCCGTGC

TATCAGATCZ

TCACACGGCG

ACATGTTAAT

GCGGATTGTG

TGGCTCGACA

CTATAAGCTA

GGAGACAGAT

GGGCTTCAGT

AGCCTATAAA

CGTTTGGGTC

CTAAGCTTTT

CGCAAGGGAT

ATATTATCGA

TTGTCGATAC

TCAGTGAGAC

TCATC.ATGGA

TAACGGAGTT

TCGTTGGACT

GCACACTCAA

CGGAGGACAC

CGTTTGCCAT

GGTTTTTGGA.

GGTCGGGTCG

TGGCTCATAT

TCGACATGAA

CAACCACGCG

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 6

I

CCTCATCTGT

AAAACTGAAA

TGTATACACG

TTCTTTAATG

AAGTAATTTA

(2) Met 1 Th r GCTCTTTAAG TTTATGGLTTC GAGTCACGTG GCAGGGGGTT TGGTATGGTi 162 AGTTTGAAGT TGCCCTCATC GAGGATTTGT GGATGTCATA TGTATGTATG 168 TGTGTTCTGA TGAAAACAGA TTTGGCTCTT TGTTTGCCCT TTTTTTTTTT 174 GGGATTTTCC TTGAATGAAA TGTAACAGTA AAAATAAGAT TTTTTTCAAT 10 GCATGTTGCA AAAAAAAAA AAAAAAAA 183 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 520 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Glu Ser Ser Ile Ser Gin Thr Leu Ser Lys Leu Ser Asp Pro Thr 5 10 Ser Leu Val Ile Val Val Set Leu Phe Lie Phe Ile Ser Phe Ile .0 0 0 0 8 Thr Arg Arg Ile Ile Gly Arg Arg Pro Pro Tyr Pro Pro 40 Asn Met Leu Met Met Asp Gin Gly Pro Arg Leu Thr His Gly Trp Pro Arg Gly Leu 5

S.

S

55O5 *5 5 S S

S

Asn Leu Ala Leu His Met Lys Lys Tyr Giy Gly Leu Cys His 70 Leu Arg Met Tyr Ala Val Ser Ser Pro Leu Gin Val Ile Ser Tyr 115 Gly Pro ?he Gin Asp Ser Vai 100 Leu Thr Tyr Asp Trp Arg Gin Met 135 Arg Ala Giu Ser 150 Arg Ser Val Ser Phe Ser Asn 105 Arg Ala Asp 120 Arg Lys Val Trp Ala Ser Glu Vai Ala Arg Gin Val Arg Pro Ala Thr Ile Ala 110 Met Ala Phe Ala His Tyr 125 Cys Vai Met Lys Val Phe 130 Ser Arg Lys 145 Lys Val 155 Asp Giu Val Asp 160 Met Val 165 Phe Ala Leu Cys Asn Val Gly 170 Thr Arg Asn Ile* 185 Gly Giu Gin Ile 180 Lys Pro Ile Asn Val 175 Thr Tyr Arg Ala Ala 190 Phe Ile Arg Ile Leu Phe Gly Ser A-la Cys Giu Lys Gly Gin Asp Glu 195 200 Gin Glu ?he Ser Lys Leu Phe Gly Ala Phe Asn Val Ala Asp Phe Ile 210 215 220 Pro Tyr Phe Gly Trp Ile Asp Pro Gin Gly Ile Asn Lys Arg Leu Val 225 230 235 240 Lys Ala Arg Asn Asp Leu Aso Gly Phe Ile Asp Asp Ile Ile Asp Glu 245 250 255 His Mec Lys Lys Lys Glu Asn Gin Asn A-a Val sp Asp Gly Aso Val 260 265 270 Val Asp Thr Asp Met Val Asp Asp Leu Leu Ala Phe Tyr Ser Glu Glu 275 280 285 Ala Lys Leu Val Ser Glu Thr Ala Asp Leu Gin Asn Ser Ile Lys Leu 290 295 300 Thr Arg Asp Asn Ile Lys Ala Ile Ile Met Aso Val Met Phe Gly Gly 305 310 315 320 Thr Glu Thr Val Ala Ser Ala Ile Glu Trp Ala Leu Thr Glu Leu Leu 325 330 335 Arg Ser Pro Glu Asp Leu Lys Arg Val Gin Gin Glu Leu Ala Glu Val 340 345 350 Val Gly Leu Asp Arg Arg Val Glu Glu Ser.Asp Ile Glu Lys Leu Thr 355 360 365 Tyr Leu Lys Cys Thr Leu Lys Glu Thr Leu Arg Met His Pro Pro Ile 370 375 380 Pro Leu Leu Leu His Glu Thr Ala Glu Asp Thr Set Ile Asp Gly Phe 385 390 395 400 Phe Ile Pro Lys Lys Ser Arg Val Met Ile Asn Ala Phe Ala Ile Gly 405 410 415 Arg Asp Pro Thr Ser Trp Thr Asp Pro Asp Thr Phe Arg Pro Ser Arg 420 425 430 0Phe Leu Glu Pro Gly Val Pro Asp Phe Lys Gly Ser Asn Phe Glu Phe 000 435 440 445 .1*.Ile Pro Phe Gly Ser Gly Arg Arg Ser Cys Pro Gly Met Gin Leu Gly 450 455 460 Leu Tyr Ala Leu Asp Leu Ala Val Ala His Ile Leu His Cys Phe Thr 465 470 475 480 Trp Lys Leu Pro Asp Gly Met Lys Pro Ser Glu Leu Asp Met Asn Asp 485 490 495 Val Phe Gly Leu Thr Ala Pro Lys Ala Thr Arg Leu Phe Ala Val Pro 500 505 510 Thr Thr Arg Leu Ile Cys Ala Leu 515 520 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 5156 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear 28 *~1 (iIMOLECULE TYPE-: DNA ((genoW2.) i) SEQUENCE-: DESCR'-PT:ON': SEQ ID ,4o:3: AAGCTTATGT

AT

AGTAACAAAT

AG

TTGGTTAGCAA

GTCACTAAAT

A(

AATTAGGAAT

G%

ATAGATGGTC

CC

ATTAAAAACG

TC

TAGGGGTTAA

A

AATAGT'TTTT

T'

ATAAATTAAA

T.

TTGTTACCACA

AAATATGATA

T

GGGTGGAGAC

A

GGTACCATACA

AGTACTACTAA

GAACAGGTGGA

ATTGCGATACA

ATTGTCTTTA

TI

TTrAATTTTA

GAACTGCGTT

ATAATTAAAA

TTcAGTAATA

CTAAACCTAA

ACGAAAAAA

TTGGCTAAAT

AATATATTTG

GATAATGCGT

CAGcTATGTT

GCTCGAATTC

CTAGATTTCC

AGTTATTAAG

TTTTGCAACA

ATTACATTTC

TTTAGGAAAA

CAAGATTT7TC 7TTZCTTAT -FrG, TTTA

ATT'TTGAT

;TTTTAAAA

TTATGTTA

:ACGACCCA

GGTTAGATT

kATAAAATA rGCTTCAAA AAAATT'rAA

TTTTTTGAA

TT-TAGAATA

TGTGATTAT

.TGCACACAC

.TTGTGTCAT

.kTACAGTG'I

LGACATACAC

'TTTTTTATC

LTACATTTGI

;GTCAAAGT(

TAAAGGAC(

GTAATAAATJ'

ATGpLGCATA

GAAAAAAA

TTATTA

TTTAGCCAC

TTATATCAC

cACTAATT'r

AGTAAAATC

TTAAACTA;

TTATAGGN;

CAN7TTGTCC 3AGTTTATG,' TrCATACCA% AACCATTTTA TTCTGTATA- TTTTTTTAAA

TATACAAAAA

ATATTC'ITAA

GAAACTAATA

GACACGAAGT

TGGTAAGAAC

ATCTGATTCG

ACTAATTAGA

CTCTCCCATT

TGATCAATAT

AAXAAAAATAA

ATTGTGCAGG

AAACACCACA

GTATTAAATT

AATATCAkcAA

ATTTTTGTAT

AATTTATATT TGGAGTTC'rA TTATTCTAAT ATTAAkTTTGT CTATTATCGA

TATTTGATAT

CTTATTACCT

TTTTATTCCA

CCTCGTGAAG CCGTGACTTAl TTGAACTTTC

TCCTATGTCCG

CCTTGALGTTT

CACCCAAATC

CCTACAGAGT

TTTGTCTTAC

TTTTGTTGTC

AALTGTGTCTI

TAGTTTACAG

ATTATGCAG'

TTGTGTAACG

CACTGTATC'

TTCAAGTATT

ATTAGTATC

k ATTACTTGTA

TAATTAATA

p, ATCCAAAAGC

AAAAATCTA

N AGAGAAAAAA

CTACCTGAA

T TATTAAATAC

AAAAATGGC

T TTAGAAdTTC T-TGTTTT1AA C AATAT'TTTTG

CCAAACTAC

'A AAACC-CACTG

AAAGTCAA'I

:C GTTTGGTATA

CTATTTATI

A TTANTATATTT

ACATAATTC

k.A TTATTTCTTT

TATTTTTV~

;T TTGCAAACTT TTAAAAGAj k.C ACATTTTTT

TAT--TAACI

:aTATT'-GT

GATCACAG'

C-TGTTTAAC

AT

TTTTAGGTTG

AT

AGGCATATAT

TA

AACGAC:GATA

CTI

TCAALCTGAGC

A;

TAGCGGATAT Al TTGTTTCAA Al GAAPLTATI'TC

T

TTTTT-rAATTT

A

GATATTATTA

C

TATTGAccTT

A,

TGAAACTACAG

ATATGATC1'AG GTTCACTTC

A

GGTCCAATTT

T

CCCACTG~~

G

7 TGTTTTTAAC

T

r GTAALTCTGA"' r~ WATTGTGA'G'T r CTGTCTAAGA

I

'r CTAATTAGTA A ACCTAPLCTGA A. AGTCATGCAC G AGTTTCTGA X1 TTGTTATTA ,T CCTATACAGT ,C ATGATrCGTC .T CGTATAAGTA ;T TTTCTWA rT TTTTTTTAGG A ATAAATGATT

TTAAT-,'TT

TA AATCACGGAA

ACATAAT-A

TTTATTTC

ATATTGCA

,TTcGATTT

ATGAGCTC

~GAAACTA

:AATACTAG

GTATTATC

ETAACC~AA

3AGTTTTITA kAkAAGTAA

SCTTTGTTT

PNGTTCGCCA

AAC'TTAAAT

TGTATCGCA

GTGATATAT

GCAACGAA

AGATTGAGG

ATGTAAGTT

V

4

ACGACAAA

'GCACAGGTA

kACCTTGTGT kAAAGTCATT A.ACGTTcATc

GTTTGTTGAA

GATATATCGA

CATTTTTCAA

ATATTTATAT

TGTAATTCCA

AGTCTACAAC

AAATTATTTC

GTTATAATTG

TTTcccT.GTT.

TAGTTATGAC

.20 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 CGAAATTTTA

GGAAGAAATA

AAAGTAATAC TTAGAATCC7'

ATTAAATAA

ATCAAGA777 TAGGAAC ZA -'TTGAGCAAG GATTTAGAAG A-TTGAA:C7T~~AA:26 2160 A=IrTCATT: GATTAATTT ,3

AATTATTTTC-

ATTTAAGTAG

TACCTCTCTA

AATCGAATCC

TATCAGATCC

TCACACGGCG

ACATGTTAAT

GCGGATTGTG

TGGCTCGACA

CTATAAGCTA

GGAGACAGAT

GGGCTTCAGT

AGCTACTTCA

AAGTGAAAGT

TGTAGGTAAG

CCGGGCAGCG

AGAGTTCTCT

GATCGATCCG

TATTGACGAT TGGGGATGTT C CAAATTAGTC P

CAAAGCAATC

TGCGTTACGT

AATTAGGAAG G

TTTAATATATA

GACAAAAAAT G TTTGATTTTA T GGACGTTATG T GTTATTACGG A' ACTTGACAGA C' CAAAGAAACC C

CACTAGTATC

CATAGGACGC G GGAACCGGGC G~ TCGTAGATCG T(

CTAAA-TAATT

TXAATrG7

ATATTTTCAA

ATAATACAAA

ATCACAT CCC .1AAAAAACA

CACGACGTCT

GCGAAGGCCT

GATGGACCAA

CCATC'rCCGC

AGTCCTTCAA

TCTGACTTAC

GAGAAAAGTG

TCGTGATGAA

CATATTCACC

ALCTCATTTCT

CCTATAAACG

TTTGGGTCAG

%AGCTTTTTG

:AAGGGATAA I

~TTATCGATG

;TCGATACCG

LGTGAGACAG C LCATGGTAA TI

LATAATACTTA

TAATTTTCT A TAGAAGCAT T

GAGAGAGAAA

TAGGACGTT A TTGGAGGAA C' GCCCCGAGG A GAGTTGAAG A TAAGGATGC AC ~CGGTTTCT T( ~CCC.AACCT C LACCGGATT T( ;CCGGGTA TC AATGC7AGTG TG-AAAAAT.AT7

GAAA'ATATA

AATGTCAAAA

AAAATGGAGA

CTCAATATGG

CTTGTCATCG

C CATATC CT C

CTCACCCACC

ATGGGATTCC

GTCCAAGACA

GACCGAGCGG

TGTGTCATGA

GTGGACAAAA

PCTCTTGCTA

rCTTTCTTTA rCGGGGAGCA

:CTGCGAGAA

;AGCCI-rCAA

~CAAGCGGCTC

ACATATGAAC

CTATGGTTGA I1

:GGATCTTCAA

TATATTTCA

.TCCATTGACC

.TTTZACTAGA

GAATATTCAG

AAAGAAAGA G TATTTAATT C GGAAACGGT A rCTAAAACGG kTCCGACAT C' :CCACCGAT C4 :ATTCCCAA G r'TGGACTGA C -AAAGGGAG C iCAACTAGG G1

SCATAATATT

ATATATGTAG

GAAATGGTGT

AAAGGGACCA

ACTTTGCCTC

AGTCTTCTAT

TTGTCTCTCT

CCGGTCCACG

GTGGTTTAGC

TCCATATGTA

GCGTCTTCTC

ACATGGCTTT

AGGTGTTTAG

TGGTCCGGTC

TATATATGTG

GTATGTACTT

kATTTTTGCA

;GGACAAGAC

:GTAGCGGAT

:GTGAAGGcCC ;AAGAAGGAG GATCTTCTT C

LPAATTCCATC

LAAGCACTA G AGTTATTTT C ,GAAAGCAAC A

ATCTACAATA

TGGACTAGT G TAATTTGAT T GCGTCGGCG A GTCCAACAA G GAGA GTTG A CCTCTCCTC C' %AATCTCGT G :CGGACACG T VATTTCGAG T~ LTATACGCG C', GTAAATAAGT1 AT TTTTTCkA

GTACATATAT

CACAATTT GA

CTGACAACAT

ATCACAIACA

TTTCATCTTC

AGGTITGGCCC

CAAlTTAGCT

CGCTGTCTCA

GAACCGGCCT

CGCTCACTAC

CCGTAAAAGA

C4GTCTCTTGT

CAA.TTAAACA

TACA=TAA

CTGAcCCGCA

;AGTTCATAA

n'CATACCAT GTAATGATC kATCAAAACG C ;CTTTTTACA G LAACTTACCC G ;TCATAGTCA T TCCTAAGTT T .GATTTTAGC A ATTATGAAA C TGGATATAT T

TTTTTATTTG

TAGAGTGGG C AACTCGCCG A CTTATCTCA A TCCACGAAA C TGATGATCA A( M'AGAC CAT CC rTATACCGT TC ~TGACTTAG CC TCAAGTAc:T 2220 AAGGAAk-.:CT 2280 GGAT GAAGAA 2340 TTAT-AAAAC-C 2400 TTCAGAAAAT 2460 CTAAGCAAAC 2520 ATCAGCTTCA 2580 ATCATAGGCA 2640 AAAAAGTATG 2700 TCACCCGAGG 2760 GCAACTAXAG 2820 GGACCGTTTT 2880 GCTGAGTCAT 2940 A~ACGTTGGTA 3000 AATATGTAAA 3060 C.CAAAACAAT 3120 kCATAACTTA 3180 ;AATCTTACA 3240 kTTTCGGGTG 3300 .AGACGGATT 3360 .TGTGGATGA 3420 PTGAAGAGGC 3480 ;TGACAATAT 3540 GTTTCTTAA 3600 TTTTGTTTG 3660 TGATCTTTT 3720 TAATGAAGA 3780 TAATTCTAA 3840 ATTTTATTA 3900 .TTAACGGA 3960 kGTCGTTGG 4020 kTGC-ACACT 4080 .GCGGAGGA 4140 GCGTTTGC 4200 ;AGGTTTTT 4260 .GGGTCGGG 4320 .GTGCTCA 4380

JI~

2 7:ATATTACAT

GAATGATGTG

GCGCCTCATC

GTGAAAACTG

ATGTGTATAC

TTTTTCTTTA

AATAAGTAAT

AAAAAAAAAT

ATTGTGTCPA

ATTTTACAAG

CTAAGGTTT

GCAAATAAAT

TCATITACCTA

TGCTTCACST

T'rTGGTCTCA

TGTGC'ICTT

AAAAGTTTGA

ACGTGTGTTC

ATGGGGAT'TT

TTAGCATGTT

TTTTTTTTAG

TTAGGGGCTG

CCCAACAAAA

TATTAGTT

GTATTTTATC

AAAAAAGACA

GGAAATTACC

CGGCTCCTAA

AAGTTTATGG

AGTTGCCCTC

TGATGAAAAC

TCCTTGAATG

GCAAAGATCG

TTATTTCACC

GAAGTTCGCT

GGTCGCAGAT

ATTTTCAGTT

ATATTTATGT

GAGTGGTTTC

TGATGGGATG

AGCCACGCGG

TTCGAGTCAC

ATCGAGGATT1

AGATTTGGCT

A.AATGTAACA

ATC?1'GGATG

TTTTTCTTTT

GGTTAAGGCT

TAAAACCACA

TACTGAGTAC

TTTTGTTAT

GTTAATTTTG

AAACCAAGTG

CTTTTCGCCG

GTGGCAGGGG

TGTGGATGTC

CTTTGTTTGC

GTAAAAATAA

AGAACTTCTA

GTTCTGGTTG

AAATCAGAGT

TGATATTTAT

TATTTACTTT

AAACTCCAAA

TTTCATTAAT

AGCTCGACAT

TGCCAACCAC

GTTTGGTATG

ATATGTATGT

CC~TTTTTTT

GATTTTTTTC

CTTAAAAAAA

TATGGTTGCC

TAAAGTTATA

AAAAAATT

TTTATTTTTT

CATACAGGTT

CTCGAG

4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5156

Claims (25)

1. An isolated nucleic acid fragment encoding an enzyme that functions in a plant to alter the guaiacyl:syringyl lignin monomer ratios in the plant, the fragment selected from the group consisting of: a nucleic acid fragment encoding an enzyme having the amino acid sequence of SEQ ID NO:2; a nucleic acid fragment encoding an enzyme having an amino acid sequence of SEQ ID NO:2 encompassing amino acid substitutions, additions and deletions that do not eliminate the function of the enzyme; the nucleic acid fragment of SEQ ID NO: 1; the nucleic acid fragment of SEQ ID N0:3; and a nucleic acid fragment of SEQ ID NO: 1 or SEQ ID NO:3 encompassing base changes that do not eliminate the function of the encoded enzyme.

2. A chimeric polynucleotide causing altered guaiacyl:syringyl lignin monomer ratios in a plant cell transformed with the chimeric polynucleotide, comprising the nucleic-acid fragment of Claim 1 operably linked in either sense or antisense orientation to a regulatory sequence. S: 3. The chimeric polynucleotide of Claim 2 wherein the regulatory sequence comprises an endogenous plant promoter effective for controlling expression of the nucleic acid fragment.

4. The chimeric polynucleotide of Claim 2 or 3 wherein the nucleic acid fragment is operably linked in the sense orientation to the regulatory sequence.

5. The chimeric polynucleotide of any of Claims 2-4 wherein the regulatory sequence comprises a promoter that controls expression of an enzyme of a plant's phenylpropanoid pathway.

6. The chimeric polynucleotide of any of Claims 2-5 wherein the regulatory sequence comprises a promoter selected from the group consisting of the promoter for the caffeic acid/5-hydroxyferulic acid O-methyltransferase gene, the promoter for the hydroxylase gene, the promoter for the (hydroxy)cinnamoyl-CoA ligase gene, the promoter for the (hydroxy)cinnamoyl-CoA reductase gene, the promoter for the (hydroxy)cinnamoyl alcohol dehydrogenase gene, the promoter for the cinnamate-4-hydroxylase gene, the promoter for the p-coumarate-3-hydroxylase gene, the promoter for the phenylalanine ammonia lyase gene and the promoter for the p-coumaroyl CoA ligase gene.

7. The chimeric polynucleotide of any of Claims 2-6 wherein the regulatory sequence comprises a promoter selected from the group consisting of cauliflower mosaic virus 35S promoter, the promoter for the phenylalanine ammonia lyase gene and the promoter for the p-coumaroyl CoA ligase gene.

8. The chimeric polynucleotide of any of Claims 2-7, further comprising a second nucleic acid fragment encoding an enzyme that exhibits activity in at least one step of the phenylpropanoid pathway.

9. The chimeric polynucleotide of Claim 8, wvherein the second nucleic acid fragment is operably linked in either sense or antisense orientation to a member selected from the group consisting of the regulatory sequence and a second regulatory sequence. The chimeric polynucleotide of Claim 8, wherein the second nucleic acid fragment is operably linked in the sense orientation to a member selected from the group consisting of the regulatory sequence or a second regulatory sequence.

11. The chimeric polynucleotide of any of Claims 2-7, further comprising a second nucleic acid fragment encoding a member selected from the group consisting of a (hydroxyl)cinnamoyl alcohol dehydrogenase enzyme and an O-methyltransferase enzyme.

12. The chimeric polynucleotide of any of Claims 2-7, further comprising a second nucleic acid fragment encoding an O-methyltransferase enzyme.

13. A transformed plant having altered guaiacyl:syringyl lignin monomer ratios relative to the ratios of an untransformed plant, comprising a host plant having incorporated therein the chimeric polynucleotide of any of Claims 2-12.

14. The transformed plant of Claim 13 wherein the host plant is selected from the group consisting of alfalfa (Medicago rice (Oryza maize (Zea mays), oil seed rape (Brassica forage grasses, tobacco (Nicotiana eucalyptus (Eucalyptus pine (Pinus spruce (Picea poplar (Populus sp.) and (Arabidopsis sp.). The transformed plant of Claim 13 wherein the host plant is a tree crop.

16. A method for altering the guaiacyl:syringyl lignin monomer ratios in a plant relative to the ratios of an untransformed plant, comprising transforming a plant with the chimeric polynucleotide of any of Claims 2-12 to provide a transformed plant, wherein the transformed plant expresses the chimeric polynucleotide, and wherein the guaiacyl:syringyl lignin monomer ratio is altered in the plant.

17. The method of Claim 16, wherein said transforming comprises: transforming a cell, tissue or organ from a host plant with the chimeric polynucleotide; (ii) selecting a transformed cell, cell callus, somatic embryo, or seed which contains the chimeric polynucleotide; (iii) regenerating a whole plant from the selected transformed cell, cell callus, somatic embryo, or seed; and (iv) selecting a regenerated whole plant which has a phenotype seictici from the group consisting of accumulation of compounds derived from sinapic acid or (2) an altered syringyl lignin monomer content relative to an untransformed host plant.

18. The method of Claim 16, wherein the nucleic acid fragment is selected from the group consisting of the sequence set forth in SEQ ID NO: 1 and the sequence set forth in SEQ ID NO:3.

19. A method of altering the content or composition of lignin in a plant, comprising stably incorporating the chimeric polynucleotide of any of Claims 2-12 into the genome of the plant by transformation means whereby the incorporated chimeric polynucleotide expresses the enzyme and whereby guaiacyl:syringyl lignin monomer content or composition is altered from that of the untransformed host plant. *o* o o0 An isolated nucleic-acid fragment encoding an active plant F5H enzyme having an amino acid sequence encoded by a mature functional protein which corresponds to SEQ ID NO.:2 and wherein the amino acid sequence encompasses amino acid substitutions, additions and deletions that do not alter the function of the active plant 5FH enzyme.

21. An isolated nucleic-acid fragment selected from the group consisting of nucleic acid fragments corresponding to SEQ ID NO.:l and SEQ ID NO.:3.

22. A chimeric gene causing altered guaiacyl:syringyl lignin monomer ratios in a plant cell transformed with the chimeric gene, the chimeric gene comprising the nucleic acid fragment of claims 20 or 21 operably linked in either sense or antisense orientation to at least one suitable regulatory sequence.

23. The chimeric gene of claim 22 wherein the nucleic acid fragment is operably linked in the sense orientation to at least one suitable regulatory sequence.

24. The chimeric gene of claim 22 wherein the at least one regulatory sequence comprises a promoter selected from the group consisting of cauliflower mosaic virus is promoter, the promoter for the phenylalanine ammonia lyase gene, the promoter for the p- coumaroyl CoA ligase gene, and endogenous plant promoters capable of controlling expression of plant F5H genes.

25. A transformed plant having altered guaiacyl:syringyl lignin monomer ratios of an untransformed plant and comprising a suitable host plant and the chimeric gene of claim S 2 22.

26. The transformed plant of claim 25 wherein the syringyl lignin monomer content is from about 0 mol% to about 95 mol% as measured in whole plant tissue.

27. The transformed plant of claim 26 wherein the suitable host plant is selected from the group consisting of alfalfa (Medicago rice (Oryza maize (Zea mays), oil seed rape (Brassica forage grasses, Arabidopsis sp., tobacco (Nicotiana sp.) and tree crops such as eucalyptus (Eucalyptus pine (Pinus spruce (Picea sp.) and poplar (Populus sp.).

28. A method of altering the activity of F5H enzyme in a plant, comprising: transforming a cell, tissue or organ from a suitable host plant with the chimeric gene of claim 22 wherein the chimeric gene is expressed; (ii) selecting transformed cells, cell callus, somatic embryos, or seeds which contain the chimeric gene; (iii) regenerating whole plants from the transformed cells, cell callus, somatic embryos, or seeds selected in step (ii); (iv) selecting whole plants regenerated in step (iii) which have a phenotype characterized by an ability of the whole plant to accumulate compounds derived from [R:\LIBA]03169.doc:tlt sinapic acid or an altered syringyl lignin monomer content relative to an untransformed host plant.

29. A method of altering the content or composition of lignin in a plant, comprising stably incorporating the chimeric gene of claim 3 into the genome of the host plant by transformation means whereby the incorporated chimeric gene expresses F5H enzyme and whereby guaiacyl:syringyl lignin monomer ratios are altered from those of the untransformed host plant. Dated 24 May, 2000 Purdue Research Foundation in Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON eq a** 4. C 36 R:\LIBA]03169.doc:tlt