# Patent

## Title
Polynucleotides encoding lignin biosynthetic pathway enzymes in coffee

## Abstract

Polynucleotides encoding polypeptides that comprise the biosynthetic pathway for lignins in the coffee plant are disclosed. Also disclosed are methods for using these polynucleotides and polypeptides for the manipulation of flavor, aroma, and other features of coffee beans, as well as the manipulation resistance to pathogen, herbivore, and insect attack in the coffee plant.

## Background
This is a U.S. National Application of International Application No. PCT/US06/040223, filed Oct. 16, 2006, which claims benefit of U.S. Provisional Application No. 60/726,812, filed Oct. 14, 2005, the entire contents of each of which are incorporated by reference herein.


FIELD OF THE INVENTION
The present invention relates to the field of agricultural biotechnology. In particular, the invention features polynucleotides from coffee plants that encode enzymes responsible for lignin synthesis, as well as methods for using these polynucleotides and polypeptides for gene regulation and manipulation of flavor, aroma and other features of coffee beans.
BACKGROUND OF THE INVENTION
Various publications, including patents, published applications and scholarly articles, are cited throughout the specification. Each of these publications is incorporated by reference herein, in its entirety. Citations not fully set forth within the specification may be found at the end of the specification.
Coffee aroma and flavor are key components in consumer preference for coffee varieties and brands. The characteristic aroma and flavor of coffee stems from a complex series of chemical reactions involving flavor precursors (Maillard reactions) that occur during the roasting of the bean. Flavor precursors include chemical compounds and biomolecules present in the green coffee bean. To date, over 800 chemicals and biomolecules have been identified as contributing to coffee flavor and aroma. (Flament, I., 2002 “Coffee Flavor Chemistry” J. Wiley U.K.). Because coffee consumers are becoming increasingly sophisticated, it is desirable to produce coffee with improved aroma and flavor in order to meet consumer preferences. Both aroma and flavor may be artificially imparted into coffee products through chemical means. See, for example, U.S. Pat. No. 4,072,761 (aroma) and U.S. Pat. No. 3,962,321 (flavor). However, to date, there is little information concerning the influence of natural coffee grain components such as polysaccharides, proteins, pigments, and lipids, on coffee aroma and flavor. One approach is to select varieties from the existing germplasm that have superior flavor characteristics. A disadvantage to this approach is that, frequently, the highest quality varieties also possess significant negative agronomics traits, such as poor yield and low resistance to diseases and environmental stresses. It is also possible to select new varieties from breeding trials in which varieties with different industrial and agronomic traits are crossed and their progeny are screened for both high quality and good agronomic performance. However, this latter approach is very time consuming, with one crossing experiment and selection over three growing seasons talking a minimum of 7-8 years. Thus, an alternative approach to enhancing coffee quality would be to use techniques of molecular biology to enhance those elements responsible for the flavor and aroma that are naturally found in the coffee bean, or to add aroma and flavor-enhancing elements that do not naturally occur in coffee beans. Genetic engineering is particularly suited to achieve these ends. For example, coffee proteins from different coffee species may be swapped. In the alternative, the expression of genes encoding naturally occurring coffee proteins that positively contribute to coffee flavor may be enhanced. Conversely, the expression of genes encoding naturally occurring coffee proteins that negatively contribute to coffee flavor may be suppressed.
Coffees from different varieties and origins exhibit significant flavor and aroma quality variations when the green grain samples are roasted and processed in the same manner. The quality differences are a manifestation of chemical and physical variations within the grain samples that result mainly from differences in growing and processing conditions, and also from differences in the genetic background of both the maternal plant and the grain. At the level of chemical composition, at least part of the flavor quality can be associated with variations in the levels of small metabolites, such as sugars, acids, phenolics, and caffeine found associated with grain from different varieties. It is accepted that there are other less well characterized flavor and flavor-precursor molecules. In addition, it is likely that structural variations within the grain also contribute to differences in coffee quality. One approach to finding new components in the coffee grain linked to coffee quality is to study the genes and proteins differentially expressed during the maturation of grain samples in different varieties that possess different quality characteristics. Similarly, genes and proteins that participate in the biosynthesis of flavor and flavor-precursor molecules may be studied.
Lignin is a phenolic polymeric material, which in angiosperms is primarily composed of three phenylpropanoid pathway-derived compounds: p-coumaroyl alcohol, coniferyl alcohol and sinapyl alcohol, i.e., the major monolignols found in plant (Hatfield R et al. 2001). These monolignols produce respectively p-hydroxyphenyl H, guaiacyl G, and syringyl S units when incorporated into the lignin polymer. Although exceptions exist, in a dicotyledonous angiosperm such as coffee, lignins consist principally of G and S units with traces of H units (Boerjan W et al. 2003). These complex polymers contribute compressive strength and increased water impermeability of the extracellular cell wall polysaccharide-protein matrix (Whetten R W et al. 1998). One response to pathogen ingression in plants is to increase the production of lignins in the cell wall, thereby reinforcing the cells surrounding the infection site and restricting further pathogen growth. (Vance C et al. 1980). Furthermore, other types of stresses, such as elevated levels of H2O2 and reduced cellulose synthesis, also result in an increased production of lignin, indicating that elevation of lignin synthesis is part of the more general stress response system in plants. (Wu G et al. 1997; and Logemann E et al. 1997).
The biosynthetic pathway for the monolignols has been controversial, with the model for the pathway changing several times in recent years (Dixon R A et al. 2001; and, Humphreys J M et al. 2002). The synthesis of lignin monomers, which is part of phenylpropanoid metabolism, begins with the deamination of phenylalanine, continues with successive hydroxylation and methylation reactions on the aromatic ring, and ends with the conversion of the side-chain carboxyl to an alcohol group (Boerjan et al. 2003). As shown in FIG. 1, the enzyme 4-hydroxycinnamoyl-CoA ligase (4CL) catalyzes an early reaction in the pathway to monolignol synthesis, the formation of the CoA esters caffeoyl-CoA, feruloyl CoA, and 5-hydroxy-feruloyl CoA (Lee et al. 1997). cDNA encoding this protein have recently been obtained and characterized from coffee, see WO 2007/044992, claiming priority to U.S. Provisional Application No. 60/726,298.
In angiosperms species, the first lignol specific enzyme identified was caffeic acid O-methyltransferase (COMT). COMT is capable of converting caffeic acid to ferulic acid, as well as converting 5-hydroxyferulic acid to sinapic acid. (Dixon et al. 2001). Down regulation of the COMT gene in maize (Zea mays) has been shown to cause a significant reduction of COMT activity (a fall of 70 to 85%), resulting in modification of lignin content and composition, and indicates that this enzyme is a key enzyme for lignin synthesis. (Piquemal J et al. 2002). Recently, the 2.2-Å crystal structure of an alfalfa COMT protein complexed with the cofactor SAH (S-adenosyl-L-homocysteine) and the substrate ferulic acid has been accomplished by Zubieta et al. (2002). This has allowed for the development of a model to explain the catalytic mechanism of COMT. This model indicates that the 3- or 5-hydroxyl group can be deprotonated by His269, facilitating the transfer of the reactive methyl group of SAM. The crystal structure of the alfalfa COMT also indicated specific residues that, a) interact in SAM recognition, b) are involved in substrate recognition, and c) are involved in various aspects of the catalytic reaction (Zubieta et al. 2002).
Ferulic acid generated by COMT can be hydroxylated by ferulate 5 hydroxylase (F5H), which is a cytochrome P450-dependent monooxygenase, to form 5-hydroxy-ferulic acid. F5H is also capable of hydroxylating coniferaldehyde and coniferyl alcohol forming 5-hydroxy-coniferaldehyde and 5-hydroxy-coniferyl alcohol respectively (Meyer K et al. 1996). F5H is believed to be potentially a rate limiting step in syringyl lignin biosynthesis, a proposal supported by the observation that an Arabidopsis mutant deficient in F5H expression is also affected at the level of sinapate esters accumulation in siliques and seeds (Ruegger M et al. 1999). All the products of F5H are also substrates for a second O-methylation catalyzed by COMT1 (FIG. 1).
CCoAOMT is a bifunctional enzyme which converts caffeoyl CoA to feruloyl CoA and 5-hydroxy-feruloyl CoA to sinapyl CoA (Inoue et al. 1998), and a CcOAOMT has been directly shown to be involved in lignin biosynthesis in the differential tracheary elements of Zinnia elegans (Ye et al. 1995). cDNA encoding CCoAOMT proteins have also been isolated and characterized from coffee, see WO 2007/044992, claiming priority to U.S. Provisional Application No. 60/726,298.
Another enzyme specifically involved in lignol biosynthesis is cinnamoyl-CoA reductase (CCR), and this enzyme catalyzes the conversion of feruloyl CoA and 5-hydroxy-feruloyl CoA into coniferaldehyde and 5-hydroxy-coniferaldehyde respectively, leading directly into the biosynthesis of G (coniferaldehyde) and S (5-hydroxy-coniferaldehyde) lignin units (Ma et al. 2005). In tobacco, down regulation of the CCR gene using an antisense construct generated plants with abnormal development and reduced growth, as well as abnormal leaf morphology and collapsed vessels. There was also an associated reduction in the level of G lignin compounds (Ralph J et al. 1998). One of the last enzymes involved in the monolignol pathway is cinnamyl alcohol dehydrogenase (CAD), which catalyzes the NADPH dependent conversion of coniferaldehyde, 5-hydroxy-coniferaldehyde and sinapaldehyde to the corresponding alcohols (Kim S J et al. 2004). In Arabidopsis, single mutants of the CAD genes AtCAD-C and AtCAD-D were found to have lower CAD activities, while a the double mutant obtained by crossing the two mutants had a 40% decrease in stem lignin content, demonstrating that these are the main CAD genes involved in stem lignin synthesis (Sibout R et al. 2005). This latter data indicates that altering a late step in lignol synthesis (i.e., altered CAD expression/activity), can be useful to influence the types of lignin generated, as well as the quantity of lignin formed.
There is little information in the literature concerning the levels of lignin in mature green coffee grain. Previously, it has been suggested that coffee grain had a lignin content of approximately 5% (Dart, S. and Nursten, H. 1985 Volatile components. In Coffee, Volumne 1; Chemistry, ed Clarke, R. and Macrae, R. Elsevier Applied Science, London, p 223-265). More recently, a compositional analysis of green grain has indicated that the carbohydrates, fat and protein made up 72% of the grain, leaving 28% of the grain corresponding to chlorogenic acids, minerals, lignin, amino acids, trigonelline, caffeine, and other compounds (Oosterveld, A., Harmsen, J., Voragen, A. and Schols, H. 2003 Extraction and characterization of polysaccharides from green and roasted C. arabica beans. Carbohydrate Polymers, 52, 285-296). From this latter data, it can be estimated that approximately 5-8% of the green grain is lignin. Other evidence for presence of significant amounts of lignin in the secondary cell walls of coffee grain cells was obtained by several different staining techniques and the use of light and transmission electron microscopy (Dentan, E. 1985. The microscopic structure of the coffee bean. In Coffee botany, biochemistry, and production of beans and beverage. Eds Clifford, M. and Willson, K. Croom Helm, London).
The lignin of the coffee grain is presumably involved in the maintenance of cellular structure, especially in the secondary cell walls of the grain, and likely also contributes to stress and insect resistance. In addition to being important for the overall health and structure of the coffee grain, it is likely that coffee grain quality can be influenced by the quantity, type, and structure of the lignin present. Lignin monomers and polymers may be directly involved in some of the chemical reactions that form coffee aromas/flavors and those that cause protein and polysaccharide degradation in the green coffee grain during coffee roasting. For example, lignin is believed to be a participant in the Maillard reaction, and potentially contributes to the generation of phenylpropanoid-derived aroma molecules such as guaiacol and 4-vinyl guaiacol. (Yeretzian C et al. 2002; and Logmann; Sagehashi, M. Miyasaka, N. Shishido, H., and Sakoda, A. 2005, Bioresource Technol. in press).
Lignins are also likely to be involved in melanoidin formation in coffee, and hence contribute to the overall antioxidant capability of this fraction. (Delgado-Andrade C et al. 2005). Lignin structure and/or quantity could also affect coffee quality indirectly by its influence on grain properties like water permeability and cell wall structure, thereby influencing, for example, the rate of water loss and the grain heating profile during coffee roasting, as well as the capability of the grain to trap volatile gases formed within the coffee endosperm during roasting (Yeretzian C et al. 2002).
Interestingly, it is believed that one or more coffee genes involved in lignin synthesis described herein is involved in the synthesis of coffee flavor molecules, or currently unknown flavor precursor molecules, in a similar fashion to that demonstrated recently in strawberry. Strawberries contain an unusual group of aroma compounds related to 2,5-dimethyl-3(2H)-furanone (DMMF). This particular compound is generated from 2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF) via an S-adenosyl-L-methionine dependent O-methyltransferase FaOMT with very high homology to the lignin synthesis enzyme COMT. The expression pattern of the FaOMT, and the enzymatic activity in the different stages of fruit ripening, suggests that FaOMT is also involved in lignin formation within the achenes and the vascular bundles of the expanding fruit, in addition to playing an important role in the biosynthesis of strawberry volatiles such as vanillin and DMMF (Weim et al. 2002).
Variety differences in lignin structure and/or quantity can also alter the extractability properties of the respective roasted grain. In maize, four brown mdrib (bm) mutants are known: bm1, which affects in CAD activity, bm2, which is associated with an over-expression of COMT, bin3a and 3b, which represent an insertion and a deletion in a COMT gene, respectively, and the bm4 mutant, which is affected in cell wall composition. Marita et al. (2003), showed that the double mutant bm1-bm2 had lower lignin content relative to the wild type. In addition, parallel reduction of esterified p-coumaroyl CoA was observed in all mutants. All observation were associated with alteration of cell wall degradability in the maize mutant (Marita J M et al. 2003).
Despite of the importance of lignin synthesis to the overall welfare of the coffee plant, as well as its probable impact on several aspects of coffee quality, at present there is no available information detailing lignin biosynthesis in coffee.
From the foregoing discussion, it will be appreciated that modulating lignin content in coffee grain by genetically modulating the production of the proteins responsible for lignin biosynthesis would be of great utility to enhance the aroma and flavor of coffee beverages and coffee products produced from such genetically engineered coffee beans. Modulating lignin content in the coffee plant also has implications for protecting the coffee plant and its fruit from pathogens, herbivores, and insects. Accordingly, a need exists to identify, isolate and utilize genes and enzymes from coffee that are involved in the biosynthesis of lignins. The present invention addresses this need.
SUMMARY OF THE INVENTION
The invention described herein features genes encoding enzymes in the lignin biosynthetic pathway in coffee plants, their encoded polypeptides, and methods for using these polynucleotides and polypeptides for gene regulation and manipulation of flavor, aroma and other features of coffee beans.
One aspect of the invention features a nucleic acid molecule isolated from coffee (Coffea spp.), having a coding sequence that encodes a lignin biosynthetic pathway enzyme. In one embodiment, the enzyme is a caffeic acid O-methyltransferase that is at least 75.4% identical to SEQ ID NO:15. In another embodiment, the enzyme is a caffeic acid O-methyltransferase that is at least 42% identical to SEQ ID NO:16. In another embodiment, the enzyme is a caffeic acid O-methyltransferase that is at least 48.1% identical to SEQ ID NO:17. In another embodiment, the enzyme is a caffeic acid O-methyltransferase that is at least 47.4% identical to SEQ ID NO:18. In another embodiment, the enzyme is a cinnamoyl CoA reductase that is at least 48.7% identical to SEQ ID NO:19. In another embodiment, the enzyme is a cinnamoyl CoA reductase that is at least 88.6% identical to SEQ ID NO:20. In another embodiment, the enzyme is a cinnamyl alcohol dehydrogenase that is at least 42.3% identical to SEQ ID NO:21. In another embodiment, the enzyme is a cinnamyl alcohol dehydrogenase that is at least 78.2% identical to SEQ ID NO:22. In another embodiment, the enzyme is a cinnamyl alcohol dehydrogenase that is at least 61.3% identical to SEQ ID NO:23. In another embodiment, the enzyme is a cinnamyl alcohol dehydrogenase that is at least 62.8% identical to SEQ ID NO:24. In another embodiment, the enzyme is a cinnamyl alcohol dehydrogenase that is at least 31.6% identical to SEQ ID NO:25. In another embodiment, the enzyme is a cinnamyl alcohol dehydrogenase that is at least 79.8% identical to SEQ ID NO:26. In another embodiment, the enzyme is a cinnamyl alcohol dehydrogenase that is at least 68% identical to SEQ ID NO:27. In another embodiment, the enzyme is a ferulate 5-hydroxylase that is at least 53% identical to SEQ ID NO:28.
In certain embodiments, the nucleic acid molecule is a gene having an open reading frame that comprises the coding sequence. Alternatively, it may comprise an mRNA molecule produced by transcription of that gene, or a cDNA molecule produced by reverse transcription of the mRNA molecule. The invention also features an oligonucleotide between 8 and 100 bases in length, which is complementary to a segment of the aforementioned nucleic acid molecule.
Another aspect of the invention features a vector comprising the above-described lignin biosynthetic pathway enzyme-encoding nucleic acid molecules. In certain embodiments, the vector is an expression vector selected from the group of vectors consisting of plasmid, phagemid, cosmid, baculovirus, bacmid, bacterial, yeast and viral vectors. In certain embodiments, the vector contains the coding sequence of the nucleic acid molecule operably linked to a constitutive promoter. In other embodiments, the coding sequence is operably linked to an inducible promoter. In other embodiments, the coding sequence of the nucleic acid molecule is operably linked to a tissue specific promoter, such as a seed specific promoter, preferably a coffee seed specific promoter.
According to another aspect of the invention, a host cell transformed with the aforementioned vector is provided. The host cell may be a plant, bacterial, fungal, insect or mammalian cell. In certain embodiments, the host cell is a plant cell selected from any one of coffee, tobacco, Arabidopsis, maize, wheat, rice, soybean barley, rye, oats, sorghum, alfalfa, clover, canola, safflower, sunflower, peanut, cacao, tomato tomatillo, potato, pepper, eggplant, sugar beet, carrot, cucumber, lettuce, pea, aster, begonia, chrysanthemum, delphinium, zinnia, and turfgrasses. The invention also features a fertile transgenic plant produced by regenerating the transformed plant cell. In a specific embodiment, the fertile transgenic plant is a Coffea species.
Another aspect of the invention features a method to modulate flavor or aroma of coffee beans. The method comprises modulating production of one or more lignin biosynthetic pathway enzymes within coffee seeds. In some embodiments, the method comprises increasing production of the one or more lignin biosynthetic pathway enzymes, e.g., by increasing expression of one or more endogenous lignin biosynthetic pathway enzyme-encoding genes within the coffee seeds, or by introducing a lignin biosynthetic pathway enzyme-encoding transgene into the plant. In other embodiments, the method comprises decreasing production of the one or more lignin biosynthetic pathway enzymes, e.g., by introducing a nucleic acid molecule into the coffee that inhibits the expression of one or more of the lignin biosynthetic pathway enzyme-encoding genes.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: Monolignols biosynthetic pathway. This representation of the plant lignins pathway is according from Hoffman et al., 2004. 4CL, 4-hydroxycinnamoyl-CoA ligase; CAD; cinnamyl-alcohol dehydrogenase; CCR, cinnamoyl-CoA reductase; COMT I, caffeic/5-hydroxyferulic acid O-methyltransferase; F5H, ferulate 5-hydroxylase; SAD, sinapyl-alcohol dehydrogenase.
FIG. 2. Protein sequence alignment of CcCOMT1, CcCOMT2p, CcCOMT3p and CcCOMT4p with plant COMT protein sequences. Alignment of putative proteins encoded by the CcCOMT genes with other COMT proteins available in the NCBI database was done using the CLUSTAL W program in the MegAlign software (Lasergene package, DNASTAR). Amino acids marked in grey match the amino acids most frequently found in this position. GenBank Accession Numbers are given in parentheses: CcCOMT IRD fruit (Coffea canephora, AAN03727) (SEQ ID NO: 29); CcCOMT IRD leaf (Coffea canephora, AAN03726) (SEQ ID NO: 30); MsCOMT (Medicago sativa, AAB46623) (SEQ ID NO: 31); NtCOMT (Nicotiana tabacum, AAL91506) (SEQ ID NO: 32); and ZeCOMT (Zinnia elegans, Q43239) (SEQ ID NO: 33). Green boxes indicate active site dimer; yellow boxes indicate catalytic residues; pink boxes show conserved residues and motifs for SAM binding; and blue boxes show active site substrate binding/positioning residues. All interactions and sites were characterized in crystal structure of alfalfa (Medicago sativa) COMT in complex with the reaction products (Zubieta et al. 2002).
FIG. 3. Protein sequence alignment of CcCCR1, and CcCCR2 with plant CCR protein sequences. Alignment of putative proteins encoded by the CcCCR genes with other CCR proteins available in the NCBI database was done using the CLUSTAL W program in the MegAlign software. Amino acids marked in grey match the amino acids most frequently found in this position. GenBank Accession Numbers are given in parentheses: EgCCR (Eucalyptus gunnii, T10735) (SEQ ID NO: 34); TaCCR (Triticum aestivumna, AAX08107) (SEQ ID NO: 35); LeCCR (Lycopersicon esculentum, AAY41880) (SEQ ID NO: 36); and StCCR1 (Solanum tuberosum, AAN71761) (SEQ ID NO: 37).
FIG. 4. Protein sequence alignment of CcCAD1ap, CcCAD1b, CcCAD2, CcCAD3, CcCAD4p, CcCAD5p, and CcCAD6p with plant CAD protein sequences. Alignment of putative proteins encoded by the CcCAD gene with other CAD proteins available in the NCBI database was done using the CLUSTAL W program in the MegAlign software. Amino acids marked in grey match the amino acids most frequently found in this position. GenBank Accession Numbers are given in parentheses: EgCAD (Eucalyptus gunnii, CAA61275) (SEQ ID NO: 38); NtCAD1 (Nicotiana tabacum, AAX15956) (SEQ ID NO: 39); and NtCAD1-1 (Nicotiana tabacum, AAX15955) (SEQ ID NO: 40).
FIG. 5. Protein sequence alignment of CcF5Hp with plant F5H protein sequences. Alignment of putative protein encoded by the CcF5Hp gene with other F5H proteins available in the NCBI database was done using the CLUSTAL W program in the MegAlign software. Amino Acids marked in grey match the amino acids most frequently found in this position. GenBank Accession Numbers are given in parentheses: AtF5H (Arabidopsis thaliana, AAD11580) (SEQ ID NO: 42); and LeF5H (Lycopersicon esculentum, AAD37433) (SEQ ID NO: 42).



DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
Definitions
Various terms relating to the biological molecules and other aspects of the present invention are used throughout the specification and claims.
The term “lignin biosynthetic pathways” refers to polypeptides that participate in lignin biosynthesis in plants, and more specifically, in coffee plants. This term encompasses the specific mechanism of action of each respective protein in the pathway. The polypeptides include without limitation, caffeic acid O-methyltransferase (“COMT”), cinnamoyl CoA reductase (“CCR”), cinnamyl alcohol dehydrogenase (“CAD”), and ferulate 5-hydroxylase (F5H), as exemplified herein.
“Isolated” means altered “by the hand of man” from the natural state. If a composition or substance occurs in nature, it has been “isolated” if it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living plant or animal is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein.
“Polynucleotide,” also referred to as “nucleic acid molecule”, generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. “Polynucleotides” include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, “polynucleotide” refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. “Polynucleotide” also embraces relatively short polynucleotides, often referred to as oligonucleotides.
“Polypeptide” refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. “Polypeptide” refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. “Polypeptides” include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from natural posttranslational processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. See, for instance, Proteins—Structure and Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York, 1993 and Wold, F., Posttranslational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York, 1983; Seifter et al., Analysis for Protein Modifications and Nonprotein Cofactors, Meth Enzymol (1990) 182:626-646 and Rattan et al., Protein Synthesis: Posttranslational Modifications and Aging, Ann NY Acad Sci (1992) 663:48-62.
“Variant” as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties. A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions or deletions in any combination. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polynucleotide or polypeptide may be naturally occurring, such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis.
In reference to mutant plants, the terms “null mutant” or “loss-of-function mutant” are used to designate an organism or genomic DNA sequence with a mutation that causes a gene product to be non-functional or largely absent. Such mutations may occur in the coding and/or regulatory regions of the gene, and may be changes of individual residues, or insertions or deletions of regions of nucleic acids. These mutations may also occur in the coding and/or regulatory regions of other genes which may regulate or control a gene and/or encoded protein, so as to cause the protein to be non-functional or largely absent.
The term “substantially the same” refers to nucleic acid or amino acid sequences having sequence variations that do not materially affect the nature of the protein (i.e., the structure, stability characteristics, substrate specificity and/or biological activity of the protein). With particular reference to nucleic acid sequences, the term “substantially the same” is intended to refer to the coding region and to conserved sequences governing expression, and refers primarily to degenerate codons encoding the same amino acid, or alternate codons encoding conservative substitute amino acids in the encoded polypeptide. With reference to amino acid sequences, the term “substantially the same” refers generally to conservative substitutions and/or variations in regions of the polypeptide not involved in determination of structure or function.
The terms “percent identical” and “percent similar” are also used herein in comparisons among amino acid and nucleic acid sequences. When referring to amino acid sequences, “identity” or “percent identical” refers to the percent of the amino acids of the subject amino acid sequence that have been matched to identical amino acids in the compared amino acid sequence by a sequence analysis program. “Percent similar” refers to the percent of the amino acids of the subject amino acid sequence that have been matched to identical or conserved amino acids. Conserved amino acids are those which differ in structure but are similar in physical properties such that the exchange of one for another would not appreciably change the tertiary structure of the resulting protein. Conservative substitutions are defined in Taylor (1986, J. Theor. Biol. 119:205). When referring to nucleic acid molecules, “percent identical” refers to the percent of the nucleotides of the subject nucleic acid sequence that have been matched to identical nucleotides by a sequence analysis program. The terms “identity” or “identical” are used interchangeably herein with the terms “homology” or “homologous.”
“Identity” and “similarity” can be readily calculated by known methods. Nucleic acid sequences and amino acid sequences can be compared using computer programs that align the similar sequences of the nucleic or amino acids and thus define the differences. In preferred methodologies, the BLAST programs (NCBI) and parameters used therein are employed, and the DNAstar system (Madison, Wis.) is used to align sequence fragments of genomic DNA sequences. However, equivalent alignments and similarity/identity assessments can be obtained through the use of any standard alignment software. For instance, the GCG Wisconsin Package version 9.1, available from the Genetics Computer Group in Madison, Wis., and the default parameters used (gap creation penalty=12, gap extension penalty=4) by that program may also be used to compare sequence identity and similarity.
“Antibodies” as used herein includes polyclonal and monoclonal antibodies, chimeric, single chain, and humanized antibodies, as well as antibody fragments (e.g., Fab, Fab′, F(ab′)2 and Fv), including the products of a Fab or other immunoglobulin expression library. With respect to antibodies, the term, “immunologically specific” or “specific” refers to antibodies that bind to one or more epitopes of a protein of interest, but which do not substantially recognize and bind other molecules in a sample containing a mixed population of antigenic biological molecules. Screening assays to determine binding specificity of an antibody are well known and routinely practiced in the art. For a comprehensive discussion of such assays, see Harlow et al. (Eds.), ANTIBODIES A LABORATORY MANUAL; Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y. (1988), Chapter 6.
The term “substantially pure” refers to a preparation comprising at least 50-60% by weight the compound of interest (e.g., nucleic acid, oligonucleotide, protein, etc.). More preferably, the preparation comprises at least 75% by weight, and most preferably 90-99% by weight, the compound of interest. Purity is measured by methods appropriate for the compound of interest (e.g., chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like).
With respect to single-stranded nucleic acid molecules, the term “specifically hybridizing” refers to the association between two single-stranded nucleic acid molecules of sufficiently complementary sequence to permit such hybridization under pre-determined conditions generally used in the art (sometimes termed “substantially complementary”). In particular, the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA or RNA molecule, to the substantial exclusion of hybridization of the oligonucleotide with single-stranded nucleic acids of non-complementary sequence.
A “coding sequence” or “coding region” refers to a nucleic acid molecule having sequence information necessary to produce a gene product, such as an amino acid or polypeptide, when the sequence is expressed. The coding sequence may comprise untranslated sequences (e.g., introns or 5′ or 3′ untranslated regions) within translated regions, or may lack such intervening untranslated sequences (e.g., as in cDNA).
“Intron” refers to polynucleotide sequences in a nucleic acid that do not code information related to protein synthesis. Such sequences are transcribed into mRNA, but are removed before translation of the mRNA into a protein.
The term “operably linked” or “operably inserted” means that the regulatory sequences necessary for expression of the coding sequence are placed in a nucleic acid molecule in the appropriate positions relative to the coding sequence so as to enable expression of the coding sequence. By way of example, a promoter is operably linked with a coding sequence when the promoter is capable of controlling the transcription or expression of that coding sequence. Coding sequences can be operably linked to promoters or regulatory sequences in a sense or antisense orientation. The term “operably linked” is sometimes applied to the arrangement of other transcription control elements (e.g., enhancers) in an expression vector.
Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
The terms “promoter,” “promoter region,” or “promoter sequence” refer generally to transcriptional regulatory regions of a gene, which may be found at the 5′ or 3′ side of the coding region, or within the coding region, or within introns. Typically, a promoter is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3′ direction) coding sequence. The typical 5′ promoter sequence is bounded at its 3′ terminus by the transcription initiation site and extends upstream (5′ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence is a transcription initiation site (conveniently defined by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
A “vector” is a replicon, such as plasmid, phage, cosmid, or virus to which another nucleic acid segment may be operably inserted so as to bring about the replication or expression of the segment.
The term “nucleic acid construct” or “DNA construct” is sometimes used to refer to a coding sequence or sequences operably linked to appropriate regulatory sequences and inserted into a vector for transforming a cell. This term may be used interchangeably with the term “transforming DNA” or “transgene.” Such a nucleic acid construct may contain a coding sequence for a gene product of interest, along with a selectable marker gene and/or a reporter gene.
A “marker gene” or “selectable marker gene” is a gene whose encoded gene product confers a feature that enables a cell containing the gene to be selected from among cells not containing the gene. Vectors used for genetic engineering typically contain one or more selectable marker genes. Types of selectable marker genes include (1) antibiotic resistance genes, (2) herbicide tolerance or resistance genes, and (3) metabolic or auxotrophic marker genes that enable transformed cells to synthesize an essential component, usually an amino acid, which the cells cannot otherwise produce.
A “reporter gene” is also a type of marker gene. It typically encodes a gene product that is assayable or detectable by standard laboratory means (e.g., enzymatic activity, fluorescence).
The term “express,” “expressed,” or “expression” of a gene refers to the biosynthesis of a gene product. The process involves transcription of the gene into mRNA and then translation of the mRNA into one or more polypeptides, and encompasses all naturally occurring post-translational modifications.
“Endogenous” refers to any constituent, for example, a gene or nucleic acid, or polypeptide, that can be found naturally within the specified organism.
A “heterologous” region of a nucleic acid construct is an identifiable segment (or segments) of the nucleic acid molecule within a larger molecule that is not found in association with the larger molecule in nature. Thus, when the heterologous region comprises a gene, the gene will usually be flanked by DNA that does not flank the genomic DNA in the genome of the source organism. In another example, a heterologous region is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein. The term “DNA construct”, as defined above, is also used to refer to a heterologous region, particularly one constructed for use in transformation of a cell.
A cell has been “transformed” or “transfected” by exogenous or heterologous DNA when such DNA has been introduced inside the cell. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA. A “clone” is a population of cells derived from a single cell or common ancestor by mitosis. A “cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.
“Grain,” “seed,” or “bean,” refers to a flowering plant's unit of reproduction, capable of developing into another such plant. As used herein, especially with respect to coffee plants, the terms are used synonymously and interchangeably.
As used herein, the term “plant” includes reference to whole plants, plant organs (e.g., leaves, stems, shoots, roots), seeds, pollen, plant cells, plant cell organelles, and progeny thereof. Parts of transgenic plants are to be understood within the scope of the invention to comprise, for example, plant cells, protoplasts, tissues, callus, embryos as well as flowers, stems, seeds, pollen, fruits, leaves, or roots originating in transgenic plants or their progeny.
Description
In one of its aspects the present invention features nucleic acid molecules from coffee that encode a variety of proteins involved in the lignin biosynthetic pathways. Representative examples of nucleic acid molecules encoding proteins that comprise the lignin biosynthetic pathways were identified from databases of over 47,000 expressed sequence tags (ESTs) from several Coffea canephora (robusta) cDNA libraries made with RNA isolated from young leaves and from the grain and pericarp tissues of cherries harvested at different stages of development. Overlapping ESTs were identified and “clustered” into unigenes (contigs) comprising complete coding sequences. The unigene sequences were annotated by performing a BLAST search of each individual sequence against the NCBI (National Center for Biotechnology Information) non-redundant protein database.
BLAST searches of the coffee EST databases using biochemically characterized protein sequences from public databases revealed gene sequences representing several important enzymes of the lignin biosynthetic pathways in the coffee plant. The fall open reading frame of several of these sequences were obtained, and a partial open reading frame was obtained for several other sequences. These cDNAs and their encoded proteins are referred to herein as follows:













cDNA
encoded protein


Enzyme
(SEQ ID NO:)
(SEQ ID NO:)



















Caffeic Acid O-Methyltransferase
CcCOMT1
1
CcCOMT1
15



CcCOMT2p
2
CcCOMT2p
16



CcCOMT3p
3
CcCOMT3p
17



CcCOMT4p
4
CcCOMT4p
18


Cinnamoyl CoA Reductase
CcCCR1
5
CcCCR1
19



CcCCR2
6
CcCCR2
20


Cinnamyl Alcohol Dehydrogenase
CcCAD1ap
7
CcCAD1ap
21



CcCAD1b
8
CcCAD1b
22



CcCAD2
9
CcCAD2
23



CcCAD3
10
CcCAD3
24



CcCAD4p
11
CcCAD4p
25



CcCAD5p
12
CcCAD5p
26



CcCAD6p
13
CcCAD6p
27


Ferulate 5-Hydroxylase
CcF5Hp
14
CcF5Hp
28









Although polynucleotides encoding proteins that catalyze key steps the lignin biosynthetic pathways from Coffea canephora are described and exemplified herein, this invention is intended to encompass nucleic acids and encoded proteins from other Coffea species that are sufficiently similar to be used interchangeably with the C. canephora polynucleotides and proteins for the purposes described below. Accordingly, when the term polypeptides or proteins that “comprise the lignin biosynthetic pathways” is used herein, it is intended to encompass all Coffea proteins that have the general physical, biochemical, and functional features described herein, as well as the polynucleotides that encode them.
Considered in terms of their sequences, the polynucleotides of the invention that encode proteins that comprise the lignin biosynthetic pathways include allelic variants and natural mutants of SEQ ID NOs:1-14, which are likely to be found in different varieties of C. arabica and C. canephora, and homologs of SEQ ID NOs:1-14 likely to be found in different coffee species. Because such variants and homologs are expected to possess certain differences in nucleotide and amino acid sequence, this invention provides isolated polynucleotides encoding proteins that comprise the lignin biosynthetic pathways that have at least about 30%, preferably at least about 40%, 45%, 50% or 55%, more preferably at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%. 78%, 79%, or 80%, even more preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, and even more preferably 90%, 91%, 92%, 93%, 94%, 95%, and most preferably 96%, 97%, 98% and 99% or more identity with any one of SEQ ID NOs:15-28, and comprise a nucleotide sequence having equivalent ranges of identity to any one of SEQ ID NOs:1-14. Because of the natural sequence variation likely to exist among proteins that comprise the lignin biosynthetic pathways, and the genes encoding them in different coffee varieties and species, one skilled in the art would expect to find this level of variation, while still maintaining the unique properties of the polypeptides and polynucleotides of the present invention. Such an expectation is due in part to the degeneracy of the genetic code, as well as to the known evolutionary success of conservative amino acid sequence variations, which do not appreciably alter the nature of the encoded protein. Accordingly, such variants and homologs are considered substantially the same as one another and are included within the scope of the present invention.
The gene regulatory sequences associated with genes encoding proteins that comprise the lignin biosynthetic pathways are of practical utility and are considered within the scope of the present invention. Promoters and other gene regulatory sequences of genes encoding proteins that comprise the lignin biosynthetic pathways from any coffee species may be obtained by the methods described below, and may be utilized in accordance with the present invention. Promoters and regulatory elements governing tissue specificity and temporal specificity of the expression of genes encoding proteins that comprise the lignin biosynthetic pathways may be used to advantage, alter or modify the expression of proteins that comprise the lignin biosynthetic pathways toward the goal of enhancing the flavor and aroma of coffee products produced from coffee beans comprising such modifications, among other utilities.
The following sections set forth the general procedures involved in practicing the present invention. To the extent that specific materials are mentioned, it is merely for the purpose of illustration, and is not intended to limit the invention. Unless otherwise specified, general biochemical and molecular biological procedures, such as those set forth in Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory (1989) or Ausubel et al. (eds), Current Protocols in Molecular Biology, John Wiley & Sons (2005) are used.
Nucleic Acid Molecules, Proteins and Antibodies
Nucleic acid molecules of the invention may be prepared by two general methods: (1) they may be synthesized from appropriate nucleotide triphosphates, or (2) they may be isolated from biological sources. Both methods utilize protocols well known in the art.
The availability of nucleotide sequence information, such as the cDNA having SEQ ID NOs:1-14, enables preparation of an isolated nucleic acid molecule of the invention by oligonucleotide synthesis. Synthetic oligonucleotides may be prepared by the phosphoramidite method employed in the Applied Biosystems 38A DNA Synthesizer or similar devices. The resultant construct may be purified according to methods known in the art, such as high performance liquid chromatography (HPLC). Long, double-stranded polynucleotides, such as a DNA molecule of the present invention, must be synthesized in stages, due to the size limitations inherent in current oligonucleotide synthetic methods. Thus, for example, a long double-stranded molecule may be synthesized as several smaller segments of appropriate complementarity. Complementary segments thus produced may be annealed such that each segment possesses appropriate cohesive termini for attachment of an adjacent segment. Adjacent segments may be ligated by annealing cohesive termini in the presence of DNA ligase to construct an entire long double-stranded molecule. A synthetic DNA molecule so constructed may then be cloned and amplified in an appropriate vector.
In accordance with the present invention, nucleic acids having the appropriate level sequence homology with part or all of the coding and/or regulatory regions genes encoding proteins that comprise the lignin biosynthetic pathways may be identified by using hybridization and washing conditions of appropriate stringency. It will be appreciated by those skilled in the art that the aforementioned strategy, when applied to genomic sequences, will, in addition to enabling isolation coding sequences for genes encoding proteins that comprise the lignin biosynthetic pathways, also enable isolation of promoters and other gene regulatory sequences associated with genes encoding proteins that comprise the lignin biosynthetic pathways, even though the regulatory sequences themselves may not share sufficient homology to enable suitable hybridization.
As a typical illustration, hybridizations may be performed according to the method of Sambrook et al., using a hybridization solution comprising: 5×SSC, 5×Denhardt's reagent, 1.0% SDS, 100 μg/ml denatured, fragmented salmon sperm DNA, 0.05% sodium pyrophosphate and up to 50% formamide. Hybridization is carried out at 37-42° C. for at least six hours. Following hybridization, filters are washed as follows: (1) 5 minutes at room temperature in 2×SSC and 1% SDS; (2) 15 minutes at room temperature in 2×SSC and 0.1% SDS; (3) 30 minutes-1 hour at 37° C. in 2×SSC and 0.1% SDS; (4) 2 hours at 45-55° C. in 2×SSC and 0.1% SDS, changing the solution every 30 minutes.
One common formula for calculating the stringency conditions required to achieve hybridization between nucleic acid molecules of a specified sequence homology (Sambrook et al., 1989):

Tm=81.5° C.+16.6 Log [Na+]+0.41(% G+C)−0.63(% formamide)−600/#bp in duplex

As an illustration of the above formula, using [Na+]=[0.368] and 50% formamide, with GC content of 42% and an average probe size of 200 bases, the Tm is 57° C. The Tm of a DNA duplex decreases by 1-1.5° C. with every 1% decrease in homology. Thus, targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42° C. In one embodiment, the hybridization is at 37° C. and the final wash is at 42° C.; in another embodiment the hybridization is at 42° C. and the final wash is at 50° C.; and in yet another embodiment the hybridization is at 42° C. and final wash is at 65° C., with the above hybridization and wash solutions. Conditions of high stringency include hybridization at 42° C. in the above hybridization solution and a final wash at 65° C. in 0.1×SSC and 0.1% SDS for 10 minutes.
Nucleic acids of the present invention may be maintained as DNA in any convenient cloning vector. In a preferred embodiment, clones are maintained in plasmid cloning/expression vector, such as pGEM-T (Promega Biotech, Madison, Wis.), pBluescript (Stratagene, La Jolla, Calif.), pCR4-TOPO (Invitrogen, Carlsbad, Calif.) or pET28a+ (Novagen, Madison, Wis.), all of which can be propagated in a suitable E. coli host cell.
Nucleic acid molecules of the invention include cDNA, genomic DNA, RNA, and fragments thereof which may be single-, double-, or even triple-stranded. Thus, this invention provides oligonucleotides (sense or antisense strands of DNA or RNA) having sequences capable of hybridizing with at least one sequence of a nucleic acid molecule of the present invention. Such oligonucleotides are useful as probes for detecting genes encoding proteins that comprise the lignin biosynthetic pathways or mRNA in test samples of plant tissue, e.g., by PCR amplification, or for the positive or negative regulation of expression genes encoding proteins that comprise the lignin biosynthetic pathways at or before translation of the mRNA into proteins. Methods in which oligonucleotides or polynucleotides may be utilized as probes for such assays include, but are not limited to: (1) in situ hybridization; (2) Southern hybridization (3) northern hybridization; and (4) assorted amplification reactions such as polymerase chain reactions (PCR, including RT-PCR) and ligase chain reaction (LCR).
Polypeptides encoded by nucleic acids of the invention may be prepared in a variety of ways, according to known methods. If produced in situ the polypeptides may be purified from appropriate sources, e.g., seeds, pericarps, or other plant parts.
Alternatively, the availability of nucleic acid molecules encoding the polypeptides enables production of the proteins using in vitro expression methods known in the art. For example, a cDNA or gene may be cloned into an appropriate in vitro transcription vector, such a pSP64 or pSP65 for in vitro transcription, followed by cell-free translation in a suitable cell-free translation system, such as wheat germ or rabbit reticulocytes. In vitro transcription and translation systems are commercially available, e.g., from Promega Biotech, Madison, Wis., BRL, Rockville, Md. or Invitrogen, Carlsbad, Calif.
According to a preferred embodiment, larger quantities of polypeptides that are active in the lignin biosynthetic pathways may be produced by expression in a suitable prokaryotic or eukaryotic system. For example, part or all of a DNA molecule, such as the cDNAs having SEQ ID NOs: 1-14, may be inserted into a plasmid vector adapted for expression in a bacterial cell (such as E. coli) or a yeast cell (such as Saccharomyces cerevisiae), or into a baculovirus vector for expression in an insect cell. Such vectors comprise the regulatory elements necessary for expression of the DNA in the host cell, positioned in such a manner as to permit expression of the DNA in the host cell. Such regulatory elements required for expression include promoter sequences, transcription initiation sequences and, optionally, enhancer sequences.
The polypeptides that comprise the lignin biosynthetic pathways produced by gene expression in a recombinant prokaryotic or eukaryotic system may be purified according to methods known in the art. In a preferred embodiment, a commercially available expression/secretion system can be used, whereby the recombinant protein is expressed and thereafter secreted from the host cell, to be easily purified from the surrounding medium. If expression/secretion vectors are not used, an alternative approach involves purifying the recombinant protein by affinity separation, such as by immunological interaction with antibodies that bind specifically to the recombinant protein. Such methods are commonly used by skilled practitioners.
The polypeptides that comprise the lignin biosynthetic pathways of the invention, prepared by the aforementioned methods, may be analyzed according to standard procedures.
Polypeptides that comprise the lignin biosynthetic pathways purified from coffee, or produced recombinantly, may be used to generate polyclonal or monoclonal antibodies, antibody fragments or derivatives as defined herein, according to known methods. Antibodies that recognize and bind fragments of the polypeptides that comprise the lignin biosynthetic pathways of the invention are also contemplated, provided that the antibodies are specific for polypeptides that comprise the lignin biosynthetic pathways. For example, if analyses of the proteins or Southern and cloning analyses (see below) indicate that the cloned genes belongs to a multigene family, then member-specific antibodies made to synthetic peptides corresponding to nonconserved regions of the protein can be generated.
Kits comprising an antibody of the invention for any of the purposes described herein are also included within the scope of the invention. In general, such a kit includes a control antigen for which the antibody is immunospecific.
Vectors, Cells, Tissues and Plants
Also featured in accordance with the present invention are vectors and kits for producing transgenic host cells that contain a polynucleotide encoding polypeptides that comprise the lignin biosynthetic pathways, or an oligonucleotide, or homolog, analog or variant thereof in a sense or antisense orientation, or a reporter gene and other constructs under control of cell or tissue-specific promoters and other regulatory sequences. Suitable host cells include, but are not limited to, plant cells, bacterial cells, yeast and other fungal cells, insect cells and mammalian cells. Vectors for transforming a wide variety of these host cells are well known to those of skill in the art. They include, but are not limited to, plasmids, phagemids, cosmids, baculoviruses, bacmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), as well as other bacterial, yeast and viral vectors. Typically, kits for producing transgenic host cells will contain one or more appropriate vectors and instructions for producing the transgenic cells using the vector. Kits may further include one or more additional components, such as culture media for culturing the cells, reagents for performing transformation of the cells and reagents for testing the transgenic cells for gene expression, to name a few.
The present invention includes transgenic plants comprising one or more copies of a gene encoding a polypeptide that comprises the lignin biosynthetic pathways, or nucleic acid sequences that inhibit the production or function of a plant's endogenous polypeptides that comprise the lignin biosynthetic pathways. This is accomplished by transforming plant cells with a transgene that comprises part of all of a coding sequence for a polypeptide that comprises the lignin biosynthetic pathways, or mutant, antisense or variant thereof, including RNA, controlled by either native or recombinant regulatory sequences, as described below. For transgenic plants, coffee species are preferred, including, without limitation, C. abeokutae, C. arabica, C. arnoldiana, C. aruwemiensis, C. bengalensis, C. canephora, C. congensis C. dewevrei, C. excelsa, C. eugenioides, and C. heterocalyx, C. kapakata, C. khasiana, C. liberica, C. moloundou, C. rasemosa, C. salvatrix, C. sessiflora, C. stenophylla, C. travencorensis, C. wightiana and C. zanguebariae. Plants of any species are also included in the invention; these include, but are not limited to, tobacco, Arabidopsis and other “laboratory-friendly” species, cereal crops such as maize, wheat, rice, soybean barley, rye, oats, sorghum, alfalfa, clover and the like, oil-producing plants such as canola, safflower, sunflower, peanut, cacao and the like, vegetable crops such as tomato tomatillo, potato, pepper, eggplant, sugar beet, carrot, cucumber, lettuce, pea and the like, horticultural plants such as aster, begonia, chrysanthemum, delphinium, petunia, zinnia, lawn and turfgrasses and the like.
Transgenic plants can be generated using standard plant transformation methods known to those skilled in the art. These include, but are not limited to, Agrobacterium vectors, polyethylene glycol treatment of protoplasts, biolistic DNA delivery, UV laser microbeam, gemini virus vectors or other plant viral vectors, calcium phosphate treatment of protoplasts, electroporation of isolated protoplasts, agitation of cell suspensions in solution with microbeads coated with the transforming DNA, agitation of cell suspension in solution with silicon fibers coated with transforming DNA, direct DNA uptake, liposome-mediated DNA uptake, and the like. Such methods have been published in the art. See, e.g., Methods for Plant Molecular Biology (Weissbach & Weissbach, eds., 1988); Methods in Plant Molecular Biology (Schuler & Zielinski, eds., 1989); Plant Molecular Biology Manual (Gelvin, Schilperoort, Verma, eds., 1993); and Methods in Plant Molecular Biology—A Laboratory Manual (Maliga, Klessig, Cashmore, Gruissem & Varner, eds., 1994).
The method of transformation depends upon the plant to be transformed. Agrobacterium vectors are often used to transform dicot species. Agrobacterium binary vectors include, but are not limited to, BIN19 and derivatives thereof, the pBI vector series, and binary vectors pGA482, pGA492, pLH7000 (GenBank Accession AY234330) and any suitable one of the pCAMBIA vectors (derived from the pPZP vectors constructed by Hajdukiewicz, Svab & Maliga, (1994) Plant Mol Biol 25: 989-994, available from CAMBIA, GPO Box 3200, Canberra ACT 2601, Australia or via the worldwide web at CAMBIA.org). For transformation of monocot species, biolistic bombardment with particles coated with transforming DNA and silicon fibers coated with transforming DNA are often useful for nuclear transformation. Alternatively, Agrobacterium “superbinary” vectors have been used successfully for the transformation of rice, maize and various other monocot species.
DNA constructs for transforming a selected plant comprise a coding sequence of interest operably linked to appropriate 5′ regulatory sequences (e.g., promoters and translational regulatory sequences) and 3′ regulatory sequences (e.g., terminators). In a preferred embodiment, a coding sequence encoding a polypeptide that comprises the lignin biosynthetic pathways under control of its natural 5′ and 3′ regulatory elements is utilized. In other embodiments, coding and regulatory sequences are swapped to alter the protein content of the seed of the transformed plant for a phenotypic improvement, e.g., in flavor, aroma or other feature.
In an alternative embodiment, the coding region of the gene is placed under a powerful constitutive promoter, such as the Cauliflower Mosaic Virus (CaMV) 35S promoter or the figwort mosaic virus 35S promoter. Other constitutive promoters contemplated for use in the present invention include, but are not limited to: T-DNA mannopine synthetase, nopaline synthase and octopine synthase promoters. In other embodiments, a strong monocot promoter is used, for example, the maize ubiquitin promoter, the rice actin promoter or the rice tubulin promoter (Jeon et al., Plant Physiology. 123: 1005-14, 2000).
Transgenic plants with coding sequences to express polypeptides that comprise the lignin biosynthetic pathways under an inducible promoter are also contemplated to be within the scope of the present invention. Inducible plant promoters include the tetracycline repressor/operator controlled promoter, the heat shock gene promoters, stress (e.g., wounding)-induced promoters, defense responsive gene promoters (e.g. phenylalanine ammonia lyase genes), wound induced gene promoters (e.g., hydroxyproline rich cell wall protein genes), chemically-inducible gene promoters (e.g., nitrate reductase genes, glucanase genes, chitinase genes, etc.) and dark-inducible gene promoters (e.g., asparagine synthetase gene) to name only a few.
Tissue specific and development-specific promoters are also contemplated for use in the present invention. Non-limiting examples of seed-specific promoters include Cim1 (cytokinin-induced message), cZ19B1 (maize 19 kDa zein), mi1ps (myo-inositol-1-phosphate synthase), and celA (cellulose synthase) (U.S. application Ser. No. 09/377,648), bean beta.-phaseolin, napin, beta.-conglycinin, soybean lectin, cruciferin, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein, waxy, shrunken 1, shrunken 2, and globulin 1, soybean 11S legumin (Bäumlein et al., 1992), and C. canephora 11S seed storage protein (Marraccini et al., 1999, Plant Physiol. Biochem. 37: 273-282). See also WO 00/12733, where seed-preferred promoters from end1 and end2 genes are disclosed. Other Coffea seed specific promoters may also be utilized, including but not limited to the oleosin gene promoter described in commonly-owned, co-pending PCT Application No. US2006/026121, the dehydrin gene promoter described in commonly-owned, co-pending PCT Application No. US2006/026234, and the 9-cis-epoxycarotenoid dioxygenase gene promoter described in commonly-owned, co-pending PCT Application No. US2006/034402. Examples of other tissue-specific promoters include, but are not limited to: the ribulose bisphosphate carboxylase (RuBisCo) small subunit gene promoters (e.g., the coffee small subunit promoter as described by Marracini et al., 2003) or chlorophyll a/b binding protein (CAB) gene promoters for expression in photosynthetic tissue; and the root-specific glutamine synthetase gene promoters where expression in roots is desired.
The coding region is also operably linked to an appropriate 3′ regulatory sequence. In embodiments where the native 3′ regulatory sequence is not use, the nopaline synthetase polyadenylation region may be used. Other useful 3′ regulatory regions include, but are not limited to the octopine synthase polyadenylation region.
The selected coding region, under control of appropriate regulatory elements, is operably linked to a nuclear drug resistance marker, such as kanamycin resistance. Other useful selectable marker systems include genes that confer antibiotic or herbicide resistances (e.g., resistance to hygromycin, sulfonylurea, phosphinothricin, or glyphosate) or genes conferring selective growth (e.g., phosphomannose isomerase, enabling growth of plant cells on mannose). Selectable marker genes include, without limitation, genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO), dihydrofolate reductase (DHFR) and hygromycin phosphotransferase (HPT), as well as genes that confer resistance to herbicidal compounds, such as glyphosate-resistant EPSPS and/or glyphosate oxidoreducatase (GOX), Bromoxynil nitrilase (BXN) for resistance to bromoxynil, AHAS genes for resistance to imidazolinones, sulfonylurea resistance genes, and 2,4-dichlorophenoxyacetate (2,4-D) resistance genes.
In certain embodiments, promoters and other expression regulatory sequences encompassed by the present invention are operably linked to reporter genes. Reporter genes contemplated for use in the invention include, but are not limited to, genes encoding green fluorescent protein (GFP), red fluorescent protein (DsRed), Cyan Fluorescent Protein (CFP), Yellow Fluorescent Protein (YFP), Cerianthus Orange Fluorescent Protein (cOFP), alkaline phosphatase (AP), β-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase (neor, G418r) dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), lacZ (encoding α-galactosidase), and xanthine guanine phosphoribosyltransferase (XGPRT), Beta-Glucuronidase (gus), Placental Alkaline Phosphatase (PLAP), Secreted Embryonic Alkaline Phosphatase (SEAP), or Firefly or Bacterial Luciferase (LUC). As with many of the standard procedures associated with the practice of the invention, skilled artisans will be aware of additional sequences that can serve the function of a marker or reporter.
Additional sequence modifications are known in the art to enhance gene expression in a cellular host. These modifications include elimination of sequences encoding superfluous polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well-characterized sequences that may be deleterious to gene expression. Alternatively, if necessary, the G/C content of the coding sequence may be adjusted to levels average for a given coffee plant cell host, as calculated by reference to known genes expressed in a coffee plant cell. Also, when possible, the coding sequence is modified to avoid predicted hairpin secondary mRNA structures. Another alternative to enhance gene expression is to use 5′ leader sequences. Translation leader sequences are well known in the art, and include the cis-acting derivative (omega′) of the 5′ leader sequence (omega) of the tobacco mosaic virus, the 5′ leader sequences from brome mosaic virus, alfalfa mosaic virus, and turnip yellow mosaic virus.
Plants are transformed and thereafter screened for one or more properties, including the presence of the transgene product, the transgene-encoding mRNA, or an altered phenotype associated with expression of the transgene. It should be recognized that the amount of expression, as well as the tissue- and temporal-specific pattern of expression of the transgenes in transformed plants can vary depending on the position of their insertion into the nuclear genome. Such positional effects are well known in the art. For this reason, several nuclear transformants should be regenerated and tested for expression of the transgene.
Methods
The nucleic acids and polypeptides of the present invention can be used in any one of a number of methods whereby the protein products can be expressed in coffee plants in order that the proteins may play a role in protecting the coffee plant from pathogens, and from herbivore, insect, or pathogen attack, and in the enhancement of flavor and/or aroma of the coffee beverage or coffee products ultimately produced from the bean of the coffee plant expressing the protein. Similarly, the polypeptides of the invention can be used in any one of a number of methods whereby the lignins and other such phytochemical products synthesized from the polypeptides may play a role in protecting the plant from pathogens, and from herbivore or insect attack, and in the enhancement of flavor and/or aroma of the coffee beverage or coffee products ultimately produced from the bean of the coffee plant containing the lignins.
With respect to protection of the plant from attack by pathogens, herbivores, and insects, lignins strengthen the plant cell wall and impede the degradation, especially digestion, of cell wall polysaccharides, and thus act as a major line of defense for the plant. (Hatfield et al. 2001). Accordingly, the ability to manipulate production of polypeptides that comprise the biosynthetic pathway for lignins in a plant, or even to use the polynucleotides and proteins of the invention to monitor such gene expression, will enable study and manipulation of the response of the coffee plant to pathogen, herbivore, or insect attack. This knowledge will enable the generation of modified coffee plants that are better equipped against disease or devastation by pathogens, herbivores or insects.
With respect to flavor and aroma of roasted coffee grain, it is expected that the polypeptides that comprise the lignin biosynthetic pathways exert some influence on the generation of coffee flavors via the Maillard reaction that occurs during roasting, by means of the content of the proteins themselves, or the products such as lignins they produce. Proteins, and particularly protein degradation products (peptides and amino acids), represent an important group of flavor precursors (Spanier et al., 2004). Therefore, relatively abundant proteins such as those that comprise the lignin biosynthetic pathways can be expected to make some contribution to the flavor generating reactions that occur during coffee roasting. Such a contribution may stem from the concentration of the proteins themselves in the coffee bean, or the concentration of the lignins ultimately produced from the proteins. The ability to monitor (e.g., through marker-assisted breeding) or manipulate protein expression profiles for polypeptides that comprise the lignin biosynthetic pathway is provided by the polynucleotides of the present invention, in accordance with the methods described herein.
Thus, one aspect of the present invention features methods to alter the profile of polypeptides that comprise the lignin biosynthetic pathway in a plant, preferably coffee, comprising increasing or decreasing an amount or activity of one or more polypeptides that comprise the lignin biosynthetic pathway in the plant. For instance, in one embodiment of the invention, a gene encoding a polypeptide that comprises the lignin biosynthetic pathway under control of its own expression-controlling sequences is used to transform a plant for the purpose of increasing production of that polypeptide in the plant. Alternatively, a coding region for a polypeptide that comprises the lignin biosynthetic pathway is operably linked to heterologous expression controlling regions, such as constitutive or inducible promoters.
Loss-of-function (null) mutant plants may be created or selected from populations of plant mutants currently available. It will also be appreciated by those of skill in the art that mutant plant populations may also be screened for mutants that over-express a particular polypeptide that comprises the lignin biosynthetic pathway, utilizing one or more of the methods described herein. Mutant populations can be made by chemical mutagenesis, radiation mutagenesis, and transposon or T-DNA insertions, or targeting induced local lesions in genomes (TILLING, see, e.g., Henikoff et al., 2004, Plant Physiol. 135(2): 630-636; Gilchrist & Haughn, 2005, Curr. Opin. Plant Biol. 8(2): 211-215). The methods to make mutant populations are well known in the art.
The nucleic acids of the invention can be used to identify mutant polypeptides that comprise the lignin biosynthetic pathways in various plant species. In species such as maize or Arabidopsis, where transposon insertion lines are available, oligonucleotide primers can be designed to screen lines for insertions in the genes encoding polypeptides that comprise the lignin biosynthetic pathways. Through breeding, a plant line may then be developed that is heterozygous or homozygous for the interrupted gene.
A plant also may be engineered to display a phenotype similar to that seen in null mutants created by mutagenic techniques. A transgenic null mutant can be created by a expressing a mutant form of a selected polypeptide that comprises the lignin biosynthetic pathways to create a “dominant negative effect.” While not limiting the invention to any one mechanism, this mutant protein will compete with wild-type protein for interacting proteins or other cellular factors. Examples of this type of “dominant negative” effect are well known for both insect and vertebrate systems (Radke et al., 1997, Genetics 145: 163-171; Kolch et al., 1991, Nature 349: 426-428).
Another kind of transgenic null mutant can be created by inhibiting the translation of mRNA encoding the polypeptides that comprise the lignin biosynthetic pathways by “post-transcriptional gene silencing.” The gene from the species targeted for down-regulation, or a fragment thereof, may be utilized to control the production of the encoded protein. Full-length antisense molecules can be used for this purpose. Alternatively, antisense oligonucleotides targeted to specific regions of the mRNA that are critical for translation may be utilized. The use of antisense molecules to decrease expression levels of a pre-determined gene is known in the art. Antisense molecules may be provided in situ by transforming plant cells with a DNA construct which, upon transcription, produces the antisense RNA sequences. Such constructs can be designed to produce full-length or partial antisense sequences. This gene silencing effect can be enhanced by transgenically over-producing both sense and antisense RNA of the gene coding sequence so that a high amount of dsRNA is produced (for example see Waterhouse et al, 1998, PNAS 95: 13959-13964). In this regard, dsRNA containing sequences that correspond to part or all of at least one intron have been found particularly effective. In one embodiment, part or all of the coding sequence antisense strand is expressed by a transgene. In another embodiment, hybridizing sense and antisense strands of part or all of the coding sequence for polypeptides that comprise the lignin biosynthetic pathways are transgenically expressed.
In another embodiment, lignin genes may be silenced through the use of a variety of other post-transcriptional gene silencing (RNA silencing) techniques that are currently available for plant systems. RNA silencing involves the processing of double-stranded RNA (dsRNA) into small 21-28 nucleotide fragments by an RNase H-based enzyme (“Dicer” or “Dicer-like”). The cleavage products, which are siRNA (small interfering RNA) or miRNA (micro-RNA) are incorporated into protein effector complexes that regulate gene expression in a sequence-specific manner (for reviews of RNA silencing in plants, see Horiguchi, 2004, Differentiation 72: 65-73; Baulcombe, 2004, Nature 431: 356-363; Herr, 2004, Biochem. Soc. Trans. 32: 946-951).
Small interfering RNAs may be chemically synthesized or transcribed and amplified is vitro, and then delivered to the cells. Delivery may be through microinjection (Tuschl T et al, 2002), chemical transfection (Agrawal N et al., 2003), electroporation or cationic liposome-mediated transfection (Brummelkamp T R et al., 2002; Elbashir S M et al., 2002), or any other means available in the art, which will be appreciated by the skilled artisan. Alternatively, the siRNA may be expressed intracellularly by inserting DNA templates for siRNA into the cells of interest, for example, by means of a plasmid, (Tuschl T et al., 2002), and may be specifically targeted to select cells. Small interfering RNAs have been successfully introduced into plants. (Klahre U et al., 2002).
A preferred method of RNA silencing in the present invention is the use of short hairpin RNAs (shRNA). A vector containing a DNA sequence encoding for a particular desired siRNA sequence is delivered into a target cell by an common means. Once in the cell, the DNA sequence is continuously transcribed into RNA molecules that loop back on themselves and form hairpin structures through intramolecular base pairing. These hairpin structures, once processed by the cell, are equivalent to siRNA molecules and are used by the cell to mediate RNA silencing of the desired protein. Various constructs of particular utility for RNA silencing in plants are described by Horiguchi, 2004, supra. Typically, such a construct comprises a promoter, a sequence of the target gene to be silenced in the “sense” orientation, a spacer, the antisense of the target gene sequence, and a terminator.
Yet another type of synthetic null mutant can also be created by the technique of “co-suppression” (Vaucheret et al., 1998, Plant J 16(6): 651-659). Plant cells are transformed with a copy of the endogenous gene targeted for repression. In many cases, this results in the complete repression of the native gene as well as the transgene. In one embodiment, a gene encoding a polypeptide that comprises the lignin biosynthetic pathways from the plant species of interest is isolated and used to transform cells of that same species.
Mutant or transgenic plants produced by any of the foregoing methods are also featured in accordance with the present invention. Preferably, the plants are fertile, thereby being useful for breeding purposes. Thus, mutant or plants that exhibit one or more of the aforementioned desirable phenotypes can be used for plant breeding, or directly in agricultural or horticultural applications. They will also be of utility as research tools for the further elucidation of the participation of polypeptides that comprise the lignin biosynthetic pathways in flavor, aroma and other features of coffee seeds associated with pigments and photosynthesis. Plants containing one transgene or a specified mutation may also be crossed with plants containing a complementary transgene or genotype in order to produce plants with enhanced or combined phenotypes.
The present invention also features compositions and methods for producing, in a seed-preferred or seed-specific manner, any selected heterologous gene product in a plant. A coding sequence of interest is placed under control of a seed-specific coffee promoter and other appropriate regulatory sequences, to produce a seed-specific chimeric gene. The chimeric gene is introduced into a plant cell by any of the transformation methods described herein or known in the art. These chimeric genes and methods may be used to produce a variety of gene products of interest in the plant, including but not limited to: (1) detectable gene products such as GFP or GUS, as enumerated above; (2) gene products conferring an agronomic or horticultural benefit, such as those whose enzyme activities result in production of micronutrients (e.g., pro-vitamin A, also known as beta-carotene) or antioxidants (e.g., ascorbic acid, omega fatty acids, lycopene, isoprenes, terpenes); or (3) gene products for controlling pathogens or pests, such as described by Mourgues et al., (1998), TibTech 16: 203-210 or others known to be protective to plant seeds or detrimental to pathogens.
The following examples are provided to describe the invention in greater detail. The examples are intended illustrate, not to limit, the invention.
Example 1
Materials and Methods for Subsequent Examples
A genomic strategy was chosen as a first approach towards understanding lignin synthesis in coffee. This strategy was based on the recently completed Nestlé/Cornell EST (Expressed Sequence Tags) library which contains 46,914 high quality EST sequences. These sequences have been assembled in-silico into 13,175 unique DNA sequences (unigenes) representing C. canephora genes being expressed in young leaves, in developing pericarp tissues (all stages mixed), and in developing grain at several distinct stages.
The unigene set of the Nestlé/Cornell database was searched using the tblastn algorithm (Altschul et al. 1990) for coffee sequences encoding full or partial ORF's (open reading frame) for polypeptides that code for caffeic acid O-methyltransferases, cinnamoyl CoA reductases, cinnamyl alcohol dehydrogenases, and ferulate-5-hydroxylases exhibiting high similarity to biochemically characterized plant proteins present in the NCBI GenBank public databases. The longest cDNA of the unigene with the best hit for each protein sequence search was then isolated and sequenced. The in silico gene expression profile observed for each coffee gene using the complete EST database (i.e., number of ESTs found for each unigene in the different tissue libraries) is presented to give an indication about the tissues expressing each gene. It is noted, however, that when the number of ESTs found in a tissue is low (as is the case for most of the lignin genes presented), this type of expression data gives only a rough estimate of the relative expression levels in each tissue. The absence of an EST does not mean that there is no expression of this gene in that particular tissue.
DNA Sequencing. Plasmid DNA was purified using Qiagen kits according to the instructions given by the manufacturer. Plasmid DNA and PCR products were sequenced by GATC Biotech AG (Konstanz, Germany) using the dideoxy termination method (S anger et al., 1977). In some cases, the unique PCR fragments produced from the 5′ RACE and genome walking experiments were directly sequenced, without purification or cloning, using the same primers as in the PCR amplification reactions. Computer analysis was performed using Laser Gene software package (DNASTAR). Homologies with sequences in the public GenBank database were identified using BLAST programs (Altschul et al. 1990) located on a Nestlé server.
Example 2
Isolation and Characterization of Coffea cDNA Clones Encoding Caffeic Acid O-Methyltransferase (COMT)
To find cDNA encoding coffee caffeic acid O-methyltransferase, the protein sequences of biochemically-characterized COMT proteins from Medicago sativa COMT (GenBank Accession Number AAB46623 (SEQ ID NO: 31), Gowri et al. 1991) and of Zinnia elegans COMT (GenBank Accession Number Q43239 (SEQ ID NO: 33), Ye et al. 1995) were used as the query sequences for a BLAST search against the Nestlé/Cornell “unigene” set 5 using the tblastn algorithm. The first search with the M. sativa COMT protein sequence uncovered 8 unigenes: #123802 (e value=e-165), #131937 (e value=1e-72), #120178 (e value=2e-63), #128376 (e value=1e-55), #120387 (e value=1e-35), #128163 (e value=2e-28), #127201 (e value=5e-24), and #120390 (e value=1e-23). These unigenes exhibited relatively high levels of homology. The second search with COMT from Zinnia elegans (SEQ ID NO: 33) uncovered the same 8 unigenes: #123802 (e value=e-159), #131937 (e value=7e-67), #120178 (e value=2e-60), #128376 (e value=2e-54), #120387 (e value=1e-32), #128163 (e value=1e-27), #120390 (e value=2e-23) and #127201 (e value=6e-22).
Coffea canephora CcCOMT1 (full ORF). A cDNA representing the 5′ end of the unigene #123802 (pcccs46w17j22) potentially encoding a complete ORF for COMT was isolated from the 46 weeks grain library (46 weeks after flowering), and fully sequenced. The insert of pcccs46w17j22 (SEQ ID NO: 1) was found to be 1314 bp long and to encode a complete ORF sequence of 1053 bp, which was called CcCOMT1 (SEQ ID NO: 1). The deduced protein sequence (SEQ ID NO: 15) has 350 amino acids, and a predicted molecular weight of 38.26 kDa. A manually optimized alignment of the deduced protein sequence (SEQ ID NO: 15) encoded by peccs46w17j22 (SEQ ID NO: 1) was performed with the COMT protein sequences CcCOMTfruit from Coffea canephora (GenBank Accession Number AAN03727) (SEQ ID NO: 29), CcCOMTleaf from Coffea canephora (GenBank Accession Number AAN03726) (SEQ ID NO: 30), MsCOMT from Medicago sativa (GenBank Accession Number AAB46623 (SEQ ID NO: 31), biochemical and crystallographic information available (Zubieta et al., 2002)), NtCOMT form Nicotiana tabacum (GenBank Accession Number AAL91506) (SEQ ID NO: 32), and ZeCOMT from Zinnia elegans (GenBank Accession Number Q43239) (SEQ ID NO: 33).
The alignment demonstrates that CcCOMT1 protein (SEQ ID NO: 15) shares 100% and 99.5% and 75.8% and 57.4% and 75.3% identity with the protein sequences CcCOMTfruit, CcCOMTleaf, MsCOMT, NtCOMT, and ZeCOMT (SEQ ID NOs: 29, 30, 31, 32, and 33) noted above, respectively (FIG. 2), and supports the initial annotation of pcccs46w17j22 (SEQ ID NO: 1) as a C. canephora caffeic acid O-methyltransferase. Moreover, this alignment and the identity between peccs46w17j22 (SEQ ID NO: 1) and the two coffee COMT sequences: CcCOMTfruit and CcCOMTleaf suggest that those are allelic sequence.
The alignment also demonstrates that the CcCOMT1 protein (SEQ ID NO: 15) contains almost all (except one) of the amino acid residues which have been identified by Zubieta, (Zubieta et al. 2002) who determined the structure of the alfalfa caffeic acid O-methyltransferase by x-ray crystallography to a) interact in SAM recognition, b) be involved in substrate recognition, and c) be involved in catalytic reaction. This alignment data also indicates that pcccs46w17j22 (SEQ ID NO: 1) encodes a full length cDNA for a C. canephora Caffeoyl CoA O-methyltransferase.
Coffea canephora CcCOMT2p (partial ORF). A cDNA representing the 5′ end of the unigene #131937 (pcccl21n18) potentially encoding a partial ORF for COMT was isolated from the leaf library and fully sequenced. The partial sequence obtained for pcccl21n18 is 893 bp long and encodes a partial ORF sequence. The first 96 bp of this cDNA (5′ end) appears to contain an intron sequence because this sequence does not match the homologous proteins, and has no homologs in the GenBank database. The partial ORF of pcccl21n18 is 672 bp long and was called CcCOMT2p (SEQ ID NO: 2). The deduced partial protein sequence (SEQ ID NO: 16) is a polypeptide of 223 amino acids, having a predicted molecular weight of 24.66 kDa.
Based on a sequence alignment with the complete ORF of CcCOMT1 (350 aa) (SEQ ID NO: 15), it was assumed that the CcCOMT2p protein (SEQ ID NO: 16) was missing over 127 amino acids at the N-terminal end. A manually optimized alignment of the deduced protein sequence encoded by pcccl21n18 was performed with the COMT protein sequences CcCOMTfruit from Coffea canephora (GenBank Accession Number AAN03727), CcCOMTleaf from Coffea canephora (GenBank Accession Number AAN03726), MsCOMT from Medicago sativa (GenBank Accession Number AAB46623), NtCOMT form Nicotiana tabacum (GenBank Accession Number AAL91506), and ZeCOMT from Zinnia elegans (GenBank Accession Number Q43239) (SEQ ID NOs: 29, 30, 31, 32, and 33).
This alignment demonstrates that CcCOMT2p protein (SEQ ID NO: 16) shares 41.7%, 42.6%, 38.2%, 42.9%, and 38.9% identity with the protein sequences CcCOMTfruit, CcCOMTleaf, MsCOMT, NtCOMT, and ZeCOMT (SEQ ID NOs: 29, 30, 31, 32, and 33) noted above, respectively (FIG. 2), and supports the initial annotation of pcccl21n18 (SEQ ID NO: 2) as a C. canephora caffeic acid O-methyltransferase. As shown in FIG. 2, six of the characterized sites described in the crystal structure of the alfalfa COMT are different in the coffea protein sequence COMT2p. The differences are as follows: a) Four of twelve conserved amino acids that have been determined to be involved in substrate binding (Zubieta et al. 2002), Phe172, His183, Ile316 and Ile319 of MsCOMT (SEQ ID NO: 31) are replaced, respectively, by a Tyr, a Pro, a Thr and a Leu residues in CcCOMT2p (SEQ ID NO: 15), b) one of three catalytic residues (Zubieta et al. 2002), Glu297 is replaced in CcCOMT2p protein (SEQ ID NO: 2) by an Asp residue, and c) two of 14 residues involved in cofactor SAM recognition (Zubieta et al. 2002), Thr211, and Asp231 are replaced in CcCOMT2 protein (SEQ ID NO: 2), respectively, by a Leu and a Glu residue.
Coffea canephora CcCOMT3p (partial ORF). A cDNA representing the 5′ end of the unigene #120178 (pcccl28d5) potentially encoding a partial ORF for COMT was isolated from the leaf library and partially sequenced at the 5′ terminal. The partial sequence obtained for pcccl28d5 is 475 bp long and encodes a partial ORF sequence. The partial ORF of pcccl28d5 (SEQ ID NO: 3) is 309 bp long and was called CcCOMT3p (SEQ ID NO: 3). The deduced partial protein sequence (SEQ ID NO: 17) is a polypeptide of 103 amino acids, having a predicted molecular weight of 10.9 kDa.
Based on an alignment with the complete ORF of CcCOMT1 (350 aa) (SEQ ID NO: 15), it was assumed that the CcCOMT3p protein (SEQ ID NO: 17) was missing over 248 amino acids at the C-terminal end. A manually optimized alignment of the deduced protein sequence (SEQ ID NO: 17) encoded by pcccl28d5 (SEQ ID NO: 3) was performed with the COMT protein sequences CcCOMTfruit from Coffea canephora (GenBank Accession Number AAN03727), CcCOMTleaf from Coffea canephora (GenBank Accession Number AAN03726), MsCOMT from Medicago sativa (GenBank Accession Number AAB46623), NtCOMT form Nicotiana tabacum (GenBank Accession Number AAL91506), and ZeCOMT from Zinnia elegans (GenBank Accession Number Q43239) (SEQ ID NOs: 29, 30, 31, 32, and 33).
The alignment demonstrates that CcCOMT3p protein (SEQ ID NO: 17) shares 44.3%, 44.3%, 48.1%, 40.6%, and 41.5% identity with the protein sequences CcCOMTfruit, CcCOMTleaf, MsCOMT, NtCOMT, and ZeCOMT (SEQ ID NOs: 29, 30, 31, 32, and 33) noted above, respectively (FIG. 2), and supports the initial annotation of pcccl28d5 (SEQ ID NO: 3) as a C. canephora caffeic acid O-methyltransferase.
Coffea canephora CcCOMT4p (partial ORF). A cDNA representing the 5′ end of the unigene #128376 (pcccp20122) potentially encoding a partial ORF for COMT was isolated from the pericarp library and fully sequenced. The partial sequence obtained for pcccp20122 is 983 bp long and encodes a partial ORF sequence. The partial ORF of pcccp20122 is 762 bp long and was called CcCOMT4p (SEQ ID NO: 4). The deduced partial protein sequence is a polypeptide of 253 amino acids (SEQ ID NO: 18), having a predicted molecular weight of 28.20 kDa.
Based on an alignment with the complete ORF of CcCOMT1 (350 aa) (SEQ ID NO: 14), it was assumed that the CcCOMT4p protein (SEQ ID NO: 18) was missing over 97 amino acids at the N-terminal end. A manually optimized alignment of the deduced protein sequence (SEQ ID NO: 18) encoded by pcccp20122 (SEQ ID NO: 4) was performed with the COMT protein sequences CcCOMTfruit from Coffea canephora (GenBank Accession Number AAN03727), CcCOMTleaf from Coffea canephora (GenBank Accession Number AAN03726), MsCOMT from Medicago sativa (GenBank Accession Number AAB46623), NtCOMT form Nicotiana tabacum (GenBank Accession Number AAL91506), and ZeCOMT from Zinnia elegans (GenBank Accession Number Q43239) (SEQ ID NOs: 29, 30, 31, 32, and 33).
The alignment demonstrates that CcCOMT4p protein (SEQ ID NO: 18) shares 43.7%, 43.7%, 40.5%, 47.4%, and 42.4% identity with the protein sequences CcCOMTfruit, CcCOMTleaf, MsCOMT, NtCOMT, and ZeCOMT (SEQ ID NOs: 29, 30, 31, 32, and 33) noted above, respectively (FIG. 2), and supports the initial annotation of poccp20122 (SEQ ID NO: 4) as a C. canephora caffeic acid O-methyltransferase. As shown in FIG. 2, seven of the characterized sites described in the crystal structure of the alfalfa COMT are different in the Coffea protein sequence COMT4p (SEQ ID NO: 18). The differences are as follows: a) Five of twelve conserved amino acids that have been determined to be involved in the substrate binding (Zubieta et al., 2002), Leu136, Ile316, Ile319, Met320 and Asn324 of MsCOMT (SEQ ID NO: 31), are replaced respectively by a Phe, a Ala, a Val, a Val and a Tyr residues in CcCOMT4p (SEQ ID NO: 18); b) one of three catalytic residues (Zubieta et al., 2002), Glu297 is replaced in CcCOMT2p protein (SEQ ID NO: 15) by a Asp residue; and c) two of 14 residues involved in cofactor SAM recognition (Zubieta et al. 2002), Thr211, and Asp231, are replaced in CcCOMT4p protein (SEQ ID NO: 18), respectively, by a Leu and a Glu residue.
Example 3
Isolation and Characterization of a Coffea canephora cDNA Clone Encoding Cinnamoyl CoA Reductase (CCR)
To find cDNA encoding coffee Cinnamoyl CoA Reductase, the protein sequences of biochemically-characterized CCR proteins Eucalyptus gunnii CCR (GenBank Accession Number T10735 (SEQ ID NO: 34), Lacombe et al. 1997) and of Triticum aestivum CCR (GenBank Accession Number AAX08107 (SEQ ID NO: 35), Ma et al. 2005) were used as the query sequences for a BLAST search against the Nestlé/Cornell “unigene” set 5 using the tblastn algorithm. The first search with the E. gunnii CCR protein sequence uncovered 1 unigenes #129581 (e value=e-121) exhibiting relatively high levels of homology. The second search with COMT from Zinnia elegans uncovered the same unigene #129581 (e value=9e-83).
Coffea canephora CeCCR1 (full ORF). The clone A5-1232, which is highly related to Cinnamoyl CoA Reductase from E. gunnii and T. aestivum (GenBank Accession Number respectively T10735 and AAX08107) (SEQ ID NOs: 34 and 35), was found in the Tours coffee cDNA collection. The insert of pA5-1232 (SEQ ID NO: 5) was found to be 1265 bp long and to encode a complete ORF sequence of 981 bp, which was called CcCCR1 (SEQ ID NO: 5). The deduced protein sequence (SEQ ID NO: 19) is a protein of 326 amino acids, having a predicted molecular weight of 36.31 kDa.
A manually optimized alignment of the deduced protein sequence (SEQ ID NO: 19) encoded by pA5-1232 was performed with the CCR protein sequences EgCCR from E. gunnii (GenBank Accession Number T10735), TaCCR from T. aestivum (GenBank Accession Number AAX08107), LeCCR from Lycopersicon esculentum (GenBank Accession Number AAY41880), and StCCR1 from Solanum tuberosum (GenBank Accession Number AAN71761) (SEQ ID NOs: 34, 35, 36, and 37). This alignment shows that CcCCR1 protein (SEQ ID NO: 19) shares 44.4%, 41%, 48.7%, and 48.7% identity with the protein sequences EgCCR, TaCCR, LeCCR, and StCCR1 (SEQ ID NOs: 34, 35, 36, and 37) noted above, respectively (FIG. 3), and supports the initial annotation of pA5-1232 as a C. canephora Cinnamoyl CoA Reductase.
Coffea canephora CcCCR2 (full ORF). A cDNA representing the 5′ end of the unigene #129581 (pcccs46w27k2) potentially encoding a complete ORF for CCR was isolated from the 46 weeks grain library (46 weeks after flowering) and fully sequenced. The insert of pcccs46w27k2 (SEQ ID NO: 6) was found to be 1354 bp long and to encode a complete ORF sequence of 999 bp, which was called CcCCR2 (SEQ ID NO: 6). The deduced protein sequence (SEQ ID NO: 20) is a protein of 332 amino acids, having a predicted molecular weight of 36.77 kDa.
A manually optimized alignment of the deduced protein sequence (SEQ ID NO: 20) encoded by pcccs46w27k2 (SEQ ID NO: 6) was performed with the CCR protein sequences EgCCR from E. gunnii (GenBank Accession Number T10735), TaCCR from T. aestivum (GenBank Accession Number AAX08107), LeCCR from Lycopersicon esculentum (GenBank Accession Number AAY41880), and StCCR1 from Solanum tuberosum (GenBank Accession Number AAN71761) (SEQ ID NOs: 34, 35, 36, and 37). This alignment shows that CcCCR2 protein (SEQ ID NO: 6) shares 77.8%, 61.4%, 87.7%, 88.6% identity with the protein sequences EgCCR, TaCCR, LeCCR, and StCCR1 (SEQ ID NOs: 34, 35, 36, and 37) noted above, respectively (FIG. 3), and supports the initial annotation of pcccs46w27k2 as a C. canephora Cinnamoyl CoA Reductase.
Example 4
Isolation and Characterization of Coffea cDNA Clones Encoding Cinnamyl Alcohol Dehydrogenase (CAD)
To find cDNA encoding coffee cinnamyl alcohol dehydrogenase, the protein sequences of biochemically characterized CAD protein Eucalyptus gunnii CAD (GenBank Accession Number CAA61275 (SEQ ID NO: 38), Goffner et al. 1998) was used as the query sequences for a BLAST search against the Nestlë/Cornell “unigene” set 5 using the tblastn algorithm. The first search with the E. gunnii CAD protein sequence uncovered 12 unigenes: #119696 (e value=5e-74), #125019 (e value=2e-71), #119457 (e value=3e-69), #124026 (e value=6e-53), #122110 (e value=1e-47), #129581 (e value=5e-42), #122897 (e value=2e-36), #132206 (e value=3e-32), #129285 (e value=2e-28), #122851 (e value=3e-27), #121958 (e value=2e-23), and #126600 (e value=8e-20). The unigenes exhibit relatively high levels of homology.
A blast (NestleBLAST) search of these 12 DNA sequences against the NCBI Non_Redundant_Protein Bank eliminated 5 unigenes not coding for CAD proteins. The search with the unigenes #124026, #129581 and #121958 indicated that they potentially code for Cinnamoyl CoA Reductase. The search with the Unigene #122897 indicated that it potentially encodes a dihydroflavonol 4 reductase. The search with the Unigene #122851 indicated that it potentially encodes an anthocyanin reductase.
Coffea canephora CcCAD1ap (partial ORF). A cDNA representing the 5′ end of the unigene #119696 (pcccs18w7121) potentially encoding a partial ORF for CAD was isolated from the 18 weeks grain library (18 weeks after flowering) and fully sequenced. The partial sequence obtained for pcccs18w7121 is 843 bp long and encodes a partial ORF sequence. The nucleotides C at position 237 and A at position 238 appear to be an insertion sequence because this sequence does not match the sequence from homologous proteins, and they generate a shift of the ORF that generates a chimeric protein sequence with no homologous sequence in the GenBank database.
The partial ORF of pcccs18w7121 (CA insertion sequence removed) is 516 bp long and was called CcCAD1ap (SEQ ID NO: 7). The deduced partial protein sequence (SEQ ID NO: 21) reveals a polypeptide of 171 amino acids, having a predicted molecular weight of 18.94 kDa.
Based on an alignment with the complete protein sequence of EgCAD (327 aa) (SEQ ID NO: 38), it was assumed that the CcCAD1ap protein (SEQ ID NO: 21) was missing over 156 amino acids at the N-terminal end. A manually optimized alignment of the deduced protein sequence (SEQ ID NO: 21) encoded by pcccs18w7121 (SEQ ID NO: 7) was preformed with the CAD protein sequences EgCAD from Eucalyptus gunnii (GenBank Accession Number CAA61275), NtCAD1 from Nicotiana tabacum (GenBank Accession Number AAX15956), and NtCAD1-1 from Nicotiana tabacum (GenBank Accession Number AAX15955) (SEQ ID NOs: 38, 39, and 40). This alignment demonstrates that CcCAD1ap protein (SEQ ID NO: 7) shares 42.3% and 40.6% and 40.1% identity with the protein sequences EgCAD, NtCAD1, and NtCAD1-1 (SEQ ID NOs: 38, 39, and 40) noted above, respectively (FIG. 4), and supports the initial annotation of pcccs18w7121 as a C. canephora cinnamyl alcohol dehydrogenase.
Coffea canephora CcCAD1b (full ORF). A cDNA representing the 5′ end of the unigene #129285 (pcccl29e10) potentially encoding a complete ORF for CAD was isolated from the leaf library and fully sequenced. The sequence obtained for pcccl29e10 is 1457 bp long and encodes a complete ORF sequence. A nucleic alignment between pcccl29e10 and pcccs18w7121 demonstrated that the two have 98.4% identity (95.9% at protein level) in their overlapping regions, and indicates that the two clones are alleles. The complete ORF of pcccl29e10 is 975 bp long and was called CcCAD1b (SEQ ID NO: 8). The deduced protein sequence (SEQ ID NO: 22) is a protein of 324 amino acids, having a predicted molecular weight of 35.53 kDa.
A manually optimized alignment of the deduced protein sequence encoded by pcccl29e10 was performed with the CAD protein sequences EgCAD from Eucalyptus gunnii (GenBank Accession Number CAA61275), NtCAD1 from Nicotiana tabacum (GenBank Accession Number AAX15956), and NtCAD1-1 from Nicotiana tabacum (GenBank Accession Number AAX15955) (SEQ ID NOs: 38, 39, and 40). This alignment demonstrates that CcCAD1b protein (SEQ ID NO: 22) shares 78.2%, 76.1%, and 77.1% identity with the protein sequences EgCAD, NtCAD1, and NtCAD1-1 (SEQ ID NOs: 38, 39, and 40) noted above, respectively (FIG. 4), and supports the initial annotation of pcccl29e10 as a C. canephora cinnamyl alcohol dehydrogenase.
Coffea canephora CcCAD2 (full ORF). A cDNA representing the 5′ end of the unigene #125019 (pcccs46w12g16) potentially encoding a complete ORF for CAD was isolated from the 46 weeks grain library (46 weeks after flowering) and fully sequenced. The sequence obtained for pcccs46w12g16 (SEQ ID NO: 9) is 1521 bp long and encodes a complete ORF sequence. The complete ORF of pcccs46w12g16 is 981 bp long, and was called CcCAD2 (SEQ ID NO: 9). The deduced protein sequence (SEQ ID NO: 23) reveals a protein of 326 amino acids, having a predicted molecular weight of 36.08 kDa.
A manually optimized alignment of the deduced protein sequence (SEQ ID NO: 9) encoded by pcccs46w12g16 (SEQ ID NO: 9) was performed with the CAD protein sequences EgCAD from Eucalyptus gunnii (GenBank Accession Number CAA61275), NtCAD1 from Nicotiana tabacum (GenBank Accession Number AAX15956), and NtCAD1-1 from Nicotiana tabacum (GenBank Accession Number AAX15955) (SEQ ID NOs: 38, 39, and 40). The alignment demonstrates that CcCAD2 protein (SEQ ID NO: 23) shares 61.3%, 60.1%, and 56.5% identity with the protein sequences EgCAD, NtCAD1, and NtCAD1-1 (SEQ ID NOs: 38, 39, and 40) noted above, respectively (FIG. 4), and supports the initial annotation of pcccs46w12g16 as a C. canephora cinnamyl alcohol dehydrogenase.
Coffea canephora CcCAD3 (full ORF). A cDNA representing the 5′ end of the unigene #119457 (pcccp12i20) potentially encoding a complete ORF for CAD was isolated from the pericarp library and partially sequenced. A second search performed in EST Tours databank allowed revealed the same clone A5-602 in the Tours bank, which had already been fully sequenced. The sequence obtained for pA5-602 is 1309 bp long and encodes a complete ORF sequence. The complete ORF of pA5-602 is 981 bp long, and was called CcCAD3 (SEQ ID NO: 10). The deduced protein sequence (SEQ ID NO: 24) is a protein of 326 amino acids, having a predicted molecular weight of 35.74 kDa.
A manually optimized alignment of the deduced protein sequence encoded by pA5-602 was performed with the CAD protein (SEQ ID NO: 24) sequences EgCAD from Eucalyptus gunnii (GenBank Accession Number CAA61275) (SEQ ID NO: 38), NtCAD1 from Nicotiana tabacum (GenBank Accession Number AAX15956) (SEQ ID NO: 39), and NtCAD1-1 from Nicotiana tabacum (GenBank Accession Number AAX15955) (SEQ ID NO: 40). This alignment demonstrates that CcCAD3 protein (SEQ ID NO: 24) shares 62.8%, 60.1%, and 57.1% identity with the protein sequences EgCAD, NtCAD1, and NtCAD1-1 (SEQ ID NOs: 38, 39, and 40) noted above, respectively (FIG. 4), and supports the initial annotation of pA5-602 as a C. canephora cinnamyl alcohol dehydrogenase.
Coffea canephora CcCAD4p (partial ORF). A cDNA representing the 5′ end of the unigene #122110 (pcccs30w33j23) potentially encoding a partial ORF for CAD was isolated from 30 weeks grain library (30 weeks after flowering) and fully sequenced. The sequence obtained to date for pcccs30w33j23 is 716 bp long and encodes a partial ORF sequence. A blast against the NCBI_Non_Redundant_Protein Bank revealed a deletion of 120 bp in the sequence at the position 309. The deletion results in a shift of the ORF and results in a chimeric protein sequence having no homologs in the GenBank database.
The partial ORF of pcccs30w33j23 (a N have been inserted at position 309) is 554 bp long and was called CcCAD4p (SEQ ID NO: 11). The deduced partial protein sequence (SEQ ID NO: 25) is a polypeptide of 183 amino acids, having a predicted molecular weight of 20.46 kDa. Based on an alignment with the complete protein sequence of EgCAD (327 aa) (SEQ ID NO: 38), it was assumed that the CcCAD4p protein (SEQ ID NO: 25) was missing over 108 amino acids at the N-terminal end. A manually-optimized alignment of the deduced protein sequence (SEQ ID NO: 25) encoded by pcccs30w33j23 (SEQ ID NO: 11) was performed with the CAD protein sequences EgCAD from Eucalyptus gunnii (GenBank Accession Number CAA61275) (SEQ ID NO: 38), NtCAD1 from Nicotiana tabacum (GenBank Accession Number AAX15956) (SEQ ID NO: 39), and NtCAD1-1 from Nicotiana tabacum (GenBank Accession Number AAX15955) (SEQ ID NO: 40). This alignment demonstrates that CcCAD4p protein (SEQ ID NO: 25) shares 31.6%, 31.6%, and 29.8% identity with the protein sequences EgCAD, NtCAD1, and NtCAD1-1 (SEQ ID NOs: 38, 39, and 40) noted above, respectively (FIG. 4), and supports the initial annotation of pcccs30w33j23 as a C. canephora cinnamyl alcohol dehydrogenase.
Coffea canephora CcCAD5p (partial ORF). A cDNA representing the 5′ end of the unigene #132206 (pcccwc22w11c3) potentially encoding a partial ORF for CAD was isolated from the 22 weeks wholes cherries library (22 weeks after flowering) and partially sequenced in 5′ terminal. The sequence obtained for pcccwc22w11c3 sequence is 744 bp long and encodes a partial ORF sequence. A blast against the NCBI_Non_Redundant_Protein Bank revealed an intron at position 539 through the end of the partial sequence. The presence of the intron in the ORF generates a chimeric protein sequence having no homologs in the GenBank database.
The partial ORF of pcccwc22w11c3 (intron removed) is 258 bp long and was called CcCAD5p (SEQ ID NO: 12). The deduced partial protein sequence (SEQ ID NO: 26) is 86 amino acids, and has a predicted molecular weight of 9.28 kDa. Based on an alignment with the complete protein sequence of EgCAD (327 aa) (SEQ ID NO: 38), it was assumed that the CcCAD5p protein (SEQ ID NO: 26) was missing over 241 amino acids at the C terminal-end. A manually optimized alignment of the deduced protein sequence (SEQ ID NO: 26) encoded by pcccwc22w11c3 was performed with the CAD protein sequences EgCAD from Eucalyptus gunnii (GenBank Accession Number CAA61275) (SEQ ID NO: 38), NtCAD1 from Nicotiana tabacum (GenBank Accession Number AAX15956) (SEQ ID NO: 39), and NtCAD1-1 from Nicotiana tabacum (GenBank Accession Number AAX15955) (SEQ ID NO: 40). This alignment demonstrates that CcCAD5p protein (SEQ ID NO: 26) shares 78.7%, 79.8%, and 76.4% identity with the protein sequences EgCAD, NtCAD1, and NtCAD1-1 (SEQ ID NOs: 38, 38, and 40) noted above, respectively (FIG. 4), and supports the initial annotation of pcccwc22w11c3 as a C. canephora cinnamyl alcohol dehydrogenase.
Coffea canephora CcCAD6p (partial ORF). A cDNA representing the 5′ end of the unigene #126600 (pcccp6j18) potentially encoding a partial ORF for CAD was isolated from pericarp library and partially sequenced at the 5′ terminus. The sequence obtained for pcccp6j18 sequence is 697 bp long and encodes a partial ORF sequence. The partial ORF of pcccp6j18 is 664 bp long and was called CcCAD6p (SEQ ID NO: 13). The deduced partial protein sequence (SEQ ID NO: 27) is a polypeptide of 221 amino acids, having a predicted molecular weight of 24.14 kDa.
Based on an alignment with the complete protein sequence of EgCAD (327 aa) (SEQ ID NO: 38), it was assumed that the CcCAD6p protein (SEQ ID NO: 13) was missing over 106 amino acids at the C-terminal end. A manually optimized alignment of the deduced protein sequence (SEQ ID NO: 27) encoded by pcccp6j18 (SEQ ID NO: 13) was performed with the CAD protein sequences EgCAD from Eucalyptus gunnii (GenBank Accession Number CAA61275) (SEQ ID NO: 38), NtCAD1 from Nicotiana tabacum (GenBank Accession Number AAX15956) (SEQ ID NO: 39), and NtCAD1-1 from Nicotiana tabacum (GenBank Accession Number AAX15955) (SEQ ID NO: 40). This alignment demonstrates that CcCAD6p protein (SEQ ID NO: 27) shares 68.0%, 67.1%, and 62.7% identity with the protein sequences EgCAD, NtCAD1, and NtCAD1-1 (SEQ ID NOs: 38, 39, and 40) noted above, respectively (FIG. 4), and supports the initial annotation of pcccp6j18 as a C. canephora cinnamyl alcohol dehydrogenase.
Example 5
Isolation and Characterization of a Coffea canephora cDNA Clone Encoding Ferulate 5-Hydroxylase (F5H)
To find cDNA encoding coffee ferulate-5-hydroxylase, the protein sequences of biochemically-characterized F5H proteins Arabidopsis thaliana F5H (GenBank Accession Number AAD11580 (SEQ ID NO: 41), Ruegger et al. 1999) was used as the query sequences for a BLAST search against the Nestlé/Cornell unigene set 5 using the tblastn algorithm. The first search with the A. thaliana sequence uncovered 4 unigenes, #120597 (e value=2e-91), #125120 (e value=1e-90), #124806 (e value=2e-68), and #128806 (e value=7 e-65), exhibiting relatively high levels of homology.
A blast (NestleBLAST) of these 4 DNA sequences against the NCBI Non_Redundant Protein Bank eliminated 3 unigenes that do not encode a F5H protein. The search with the unigenes #120597 and #125120 indicated that they potentially encode Cinnamoyl CoA Reductase. The search with unigene #122897 indicated that it potentially encodes cytochrome P450 type protein. The search with unigene #124806 indicated that it potentially encodes hydroxylase like cytochrome P450 type protein and thus was investigated further.
Coffea canephora CcF5Hp (partial ORF). A cDNA representing the 5′ end of the unigene #128806 (pcccl18j3) that potentially encoded a partial ORF for F5H was isolated from the leaf library and fully sequenced. The insert of pcccl18j3 was found to be 934 bp long and to encode a partial ORF sequence of 654 bp, which was called CcF5Hp (SEQ ID NO: 14). The deduced protein sequence (SEQ ID NO: 28) shows a protein of 217 amino acids, having a predicted molecular weight of 24.83 kDa.
A manually optimized alignment of the deduced protein sequence (SEQ ID NO: 28) encoded by pcccl18j3 (SEQ ID NO: 14) was performed with the F5H protein sequences AtF5H from Arabidopsis thaliana (GenBank Accession Number AAD11580) (SEQ ID NO: 41), and LeF5H from Lycopersicon esculentum (GenBank Accession Number AAD37433) (SEQ ID NO: 42). This alignment demonstrates that CcF5Hp protein (SEQ ID NO: 28) shares 53% and 50% identity with the overlapping region of protein sequences AtF5H, and LeF5H (SEQ ID NOs: 41 and 42) noted above, respectively (FIG. 5), and supports the initial annotation of pcccl18j3 as a C. canephora ferulate-5-hydroxylase.
Example 6
Expression of Lignin Genes
The number of ESTs associated with a particular unigene gives an estimation of the expression level of the associated gene in each library (in each tissue). Therefore, an examination of the number of ESTs within the different unigenes of the lignin genes discussed above can give a broad overview of the expression of these genes. All the unigenes discussed herein, and the number of ESTs in each library for these unigenes, are provided in Table 1.







TABLE 1







In silico distribution of ESTs in the unigenes.









Cornell Bank in silico expression














Gene
cccl
cccp
ccc22wc
cccs18w
cccs30w
cccs42w
cccs46w





CcCOMT1






3


CcCOMT2p
1


CcCOMT3p
1

1


CcCOMT4p
1


CcCCR1


1


CcCCR2






1


CcCAD1ap



1
2

1


CcCAD1b
1


CcCAD2




2

2


CcCAD3


1


CcCAD4p




2


CcCAD5p


1


CcCAD6p

1


F5H
1





Table 1. The number of ESTs found for each gene are presented in relation to the libraries in which these ESTs were found.






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The present invention is not limited to the embodiments described and exemplified above, but is capable of variation and modification within the scope of the appended claims.









Sequence Listing:




Sequence (SEQ ID NO: 1)


<213> OrganismName: Coffea canephora


<400> PreSequenceString:


accttttcct tcgtcccgga agcttttccc ttcacctttg attcctctcc agccaaacaa
60


 


acctaccccc caggacattt ctgatttctg aagctgaagt tggagtaaaa agaaaaagaa
120


 


atcaaataat ggcagaggag gaagcttgct tattcgccat gagcctggct agtgcatccg
180


 


tccttcctat ggtactcaaa tcagccatcg aacttgacct cctggagctt atagccaagg
240


 


ctggtcctgg cgcctacgtc tccccatcgg aactcgccgc acagctcccc acccacaacc
300


 


cggaagctcc tatcatgctt gatcgcatcc tccgactcct ggccacctac tctgtcctcg
360


 


attgcaagct caacaatctg gccgatggtg gcgtcgagag gctttacggt ctggcccctg
420


 


tttgcaaatt cttgaccaag aacgctgatg gtgtgtccat ggcccctctt ttgctcatga
480


 


atcaagataa ggtcctcatg gaaagctggt atcacttaaa ggatgcggtt cttgatggag
540


 


gaatcccttt caacaaggcc tacggaatga ctgcattcga atatcacgga accgatccca
600


 


gattcaacaa ggtgtttaac cagggaatgt ctaatcactc caccattacc atgaagaaga
660


 


ttttggaagt ttacagaggg tttgagggtc tgaagacggt ggtcgacgtg ggaggtggaa
720


 


ctggggctac gctcaatatg atcatcagca aatatcccac gatcaagggc atcaactttg
780


 


agctcccgca cgtcgtagag gacgccccgt ctcattccgg ggtggagcat gtgggtgggg
840


 


atatgtttgt tagcgtccct aaaggggatg ccattttcat gaagtggatt tgccatgatt
900


 


ggagcgacga ccactgccgg aaactcttga agaactgcta ccaagcactt ccggacaacg
960


 


ggaaggtgat ccttgccgaa tgtgtccttc cggaagcccc agacacctcg ctcgctactc
1020


 


agaatgtcgt ccacgttgat gtcgtcatgt tggcccacaa ccctggtggg aaagagagga
1080


 


ctgagaagga attcgaggcc ttggcaaagg gggctggatt caaagaattc cgcaaggttt
1140


 


gctctgctgt caatacctgg atcatggagc tgtgcaaatg aaggcttact tgcttgaatg
1200


 


aaccctttgt tttgggagac gtcatttttt ctcttgcgtg attggattgg tggtttctaa
1260


 


tcagtatagc cgtctactaa tgaaccttcc ttctgtatta ctattatttc acag
1314


<212> Type: DNA


<211> Length: 1314


SequenceName: CcCOMT1 cDNA


 


Sequence (SEQ ID NO: 2)


<213> OrganismName: Coffea canephora


<400> PreSequenceString:


aaaagatata tcttgcagtt caatgggcta ctacagatca ttggttacag gcatggtgtt
60


 


tcgatgacaa agactctact tgattttcat ccaatgtatg aacttgggaa ttctttgctt
120


 


gaaggaggga ttccatttaa tagagttcat ggaatgcacg catttgacta ccctagtaga
180


 


gatcccaggt ataatgagct tttcaacaag ggaatggttg gtcctacagc cataacaatg
240


 


aaaaaattgc ttcaacaata taaaggattt gagcaccttc agacattggt tgatgttggt
300


 


ggtggtcttg gaataaccct tcacaagatt atatcaaaat acccttctat aaggggtatc
360


 


aattttgatc ttccacatgt cattgaaaac gcgccatcct atcttggagt ggaacacatt
420


 


ggtggagaca tgtttgaaag cgttcctgga ggagatgcta tttttatgaa gatgatactc
480


 


catgattgga gtgatgatca ctgcttaaag ctgctgaaga actgcttcaa agctctacca
540


 


gatcatggca aagtcatcgt tgttgatttg gttctacccg taaaacctga tactagtgcc
600


 


tttgtaaaag gcattttcca gactgatgct ctcatgatga ctcaaaatcc tggagggaaa
660


 


gagcgatcag aatctgatgt tcgggccttg gctatcagag ctggatttaa agacataaag
720


 


ttagaatgtt gtgtgggtag tcttggggtc ctggagttgt acaaatagtt atgtctgccc
780


 


atttggcagg tttttcattt gaagaaattt cttttgtttg cttgtatttc ttaaataatc
840


 


tctatttggt ctaatgagcg tagtgtttct taaaaaaaaa aaaaaaaaaa aaa
893


<212> Type: DNA


<211> Length: 893


SequenceName: CcCOMT2p partial cDNA


 


Sequence (SEQ ID NO: 3)


<213> OrganismName: Coffea canephora


<400> PreSequenceString:


cttaatcatt tcaaaatttg ccgtcaatga gtggcaatta acacaacccc cacacccaat
60


 


ccaatatatg ctacagtaca actaatctga atttatatac gtatgaccta gctgggatct
120


 


atcatcatct acagatttaa gagttaaagg tagtagcagt agtgatatgg attcttcgtc
180


 


aagagcaacc gacaatgttg ttgttgaagc agggctagat gagcaagaag agcaacactt
240


 


ctcgtacgcg atgcagctgg tcacctctgt atctttgccc atggtgctgc tggctgccat
300


 


ccggctcgac gtgttggagg tcattgccca agcgggtcca ggtgcccaat tgtcgccttg
360


 


ggacattgca gcccaggtag gtcctaaaaa cccagatgcg gctgctatgc tggatagaat
420


 


gctgcagctc ctggctagct actcggtgct cacctgctct gttgccgagg cggat
475


<212> Type: DNA


<211> Length: 475


SequenceName: CcCOMT3p partial cDNA


 


Sequence (SEQ ID NO: 4)


<213> OrganismName: Coffea canephora


<400> PreSequenceString:


caagttcttt gtacagaaca aaacaaaggg aggaggttca ctaggctccg ttctgggcct
60


 


gcttcaagat aaggtcttca ttgacagttg gtaccaatta gaagatgcag ttcgcaaagg
120


 


gggagatccg tttcacaggg cgcacggtac acatgcattt gaatttcttg gaagcgaccc
180


 


cagattcaat gaggtattca acaaggcaat gatccaccac acagctatcg tcataaacag
240


 


aatgcttgaa cggtacaaag gttttgagca cctcaaaact ttggtagatg ttggtggtgg
300


 


tcttggaatg aacctcaata taatcacaac taaataccct agtctcaagg gtattaattt
360


 


tgatttgcca catgttatac aacatgcacc agcctatcct ggtgttgaac atgttggagg
420


 


agacatgttt gaaagtgttc cacaggggga tgccattttt atgaagtgga tacttcatga
480


 


ttgggatgat ggtcattgct tgaagctgct gaaaaattgt tacaaggctt taccagacaa
540


 


tggaaaggta atagctgttg acgcaattct tcctgtggtt cctgatgata gtgcacgcga
600


 


caaagctact tgccaagcag atcttgttgt ggtgactcaa tataggggag gaattgagag
660


 


atatgaaaca gagtttcttg ccctggctac tgctgctgga tttaaaggca taagtgtgaa
720


 


atgttttgta tgtaacttgt gggtcatgga gttctataag tagatgactg gttcatgggg
780


 


cactgtactt taaaacctta aatgttgtat gttgaatgat tgccaatgtc atggtgacta
840


 


tctgcaatct acaacgagga aaaaaactgc aatattgacc tcgtaagaat aaatttatac
900


 


attgttttgt ttccgaatat gataattatg atggagttaa ggcaaaaaaa aaaaaaaaaa
960


 


aaaaaaaaaa aaaaaaaaaa aaa
983


<212> Type: DNA


<211> Length: 983


SequenceName: CcCOMT4p partial cDNA


 


Sequence (SEQ ID NO: 5)


<213> OrganismName: Coffea canephora


<400> PreSequenceString:


aagcagtggt aacaacgcag agtacgcggg gaaccaaaaa agaactaata ctcataataa
60


 


agaaaagaat aaaactttat ccaccttctt gatcaatcta tagtgaatct ggtactaaaa
120


 


tactaatccc actactgcta caagtaagca atcagacatg gcagcgtggg aggctgagaa
180


 


ggccaggaca gtatgtgtta caggagcagg gggatacctg ggatcttggt tagtcaagct
240


 


actcctttcc cgccattata ctgttcatgc caccctcaga aatcccgagg atgagaaata
300


 


tgttcatctg aagaaacttg acaaagcagc tgagaatttg aaactcttta aggctgattt
360


 


gctggattac aactccattt ctgcagccat caggggctgt gatggcgtat ttcatgtagc
420


 


tagtcctgtt ccttcaggct ctgttcccaa tcctgaggtt gaacttgttg agccggctgt
480


 


aaagggtacc cttaatgtac tgaaggcttg ttctgaagca aatgtcaagc gcgttgtagc
540


 


tgtttcctct gttgctgctg ttgttgtgag tcctaatcgg cataaaggtg aaattataga
600


 


tgagacgtgt tggtcagacg gggaatactg caagacaaca aataactggt attgttactc
660


 


caagacggtt gctgaaagtg aggctttaca atatgcaaaa gaaactggcc ttgatgtttt
720


 


aactgtatgc ccatcctttg ttctcggccc catgcttcag catgatgtga atgctagcag
780


 


tctggctctt ataaagctgt tgaaagaagg atatgaagaa atagaaaaca aattccggga
840


 


tatggtagat gtgcgtgatg tggctgaagc actgcttttg gtttacggga gacctgaagc
900


 


tgaagggcgg tacatatgtt catctcacct cactacgaca aaggatacgg tggaaattct
960


 


gaggaaaaac tatcccaact ataagtaccc taagagattt atagaggtga aggatgatca
1020


 


aggccgagga aatgttagct cagaaaaatt gcagaggctg ggctggagat ataggccagt
1080


 


ggaagaaact cttgttgact ctgtcgaaag ctaccagcag gctgggatct tggattgatc
1140


 


actttctgcc ttttctgttt ttttgttaat gggacttgtg gattattttg taaaagaatt
1200


 


tttggatcta ttaaaagtgg aatttgtgct tgtagaaaaa aaaaaaaaaa aaaaaaaaaa
1260


 


aaaaa
1265


<212> Type: DNA


<211> Length: 1265


SequenceName: CcCCR1 cDNA


 


Sequence (SEQ ID NO: 6)


<213> OrganismName: Coffea canephora


<400> PreSequenceString:


cctttgcctt tcattgcaga agaaattttg ttctcgcgcg cagaaatctt ctcattactt
60


 


accaacctca cgtctcacct aaaccaaaaa gaaaagaaaa gaaaagaaaa accaggtcag
120


 


aagcatgcct tcagtttccg gccaagtcgt ctgtgtcact ggcgccggtg gctacatcgc
180


 


ttcctggata gttaagctcc tccttgaaaa aggctacact gttagaggaa ctgttagaaa
240


 


ccctgatgat gcgaagaatg gtcacttgcg ggagctagaa ggagcaaaag agagattgac
300


 


actatgcaga gctgaccttc ttgattatca gagtttacgt gaagccatca aaggctgtga
360


 


tggggttttc cacactgctt cccccgttac ggatgatccg gaacaaatgg tggagccagc
420


 


ggtgattggg accaaaaatg tgatcaacgc agccgccgag gccaaggtcc ggcggatggt
480


 


tttcacctca tcaattggtg cggtttacat ggaccccaac agggaacctg aaaaagttgt
540


 


ggacgagagt tgttggagtg atcttgaatt ctgcaagaat actaagaatt ggtattgcta
600


 


cgggaaagct gtggcagaaa aagcagcatg ggacgaagcc aaggagaaag gggtggattt
660


 


ggtggtgatc aacccggtgc tggtgttggg accattgttg caaccgaccg tgaatgccag
720


 


tattcttcat atcctcaaat acttgactgg atctgcaaag acttatgcca actctgtgca
780


 


ggcctacgtg cacgttaagg atgttgcatt ggcacacgtt cttatctacg agactccctc
840


 


ggcatctggg agatacctct gcgccgagag cgttcttcat cgcggtgaag tggttgaaat
900


 


cttggccaaa ctctttccgg agtacccaat tccaaccaag tgctccgatg aaacaagacc
960


 


aagagcaaaa gcttacaagt tcacaaatca gaagctcaag gatttggggt ttgaattcac
1020


 


accggtgaaa cagtgcctat atgagacggt taaaagtctt caggagaagg ggcagatacc
1080


 


cctccctact cagaacgata agcccattaa aattcactac tagtctgact ttattctggt
1140


 


tgctattgat atcaaaatgt tagcttttta tcattgaaga agaagaagta gtagaagtat
1200


 


aaagaatgtt taaggttgca atccaaatcc gttgcttgtg aaccagaatg tttaaggcag
1260


 


tagtatttgt gctccagatt tccgtacccg atgtgaagtc tttactacct aaaaaaaaaa
1320


 


aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa
1354


<212> Type: DNA


<211> Length: 1354


SequenceName: CcCCR2 cDNA


 


Sequence (SEQ ID NO: 7)


<213> OrganismName: Coffea canephora


<400> PreSequenceString:


agcaaacgaa gttatggtat gttctctcaa agacattggc tgaggatgct gcctggaagt
60


 


ttgtaaaaga gaaaggttta gacatggtaa cgataaaccc agctatggtg attgggcctc
120


 


tgttacagcc aacacttaac accagtgctg ctgcaatttt gaatttaata aacggtgcag
180


 


aaacatttcc aaattcttct atgggctggg ttgatgtcaa agatgttgct aatgcacata
240


 


tactatagct tttgaaaatc catctgctag tggaagatac tgcctggtcg aaagagtggt
300


 


acactactct gaagttgtga agatcttgcg tgaaatttat ccttcctcaa aacttccaga
360


 


aaagtgcgct gatgacaagc catttgtgcc gacgtaccag gtttccaagg agaaggcaaa
420


 


aagcttaggt cttgaattca ttccccttga gcaaagcatc aaggaaacag ttgaaagctt
480


 


gaaggagaag aactttttgg actcttctgc tgcactttga atatcgcccg aaacatgatg
540


 


aggaacatcg agagttggta gcagtgccta ttgaaaatat gttttccaaa cccctatatg
600


 


taccaagcct ctctgactga cgttacatgc atccacagag atttagaatc tacatatgaa
660


 


acaataaagt actcgtcttt tttgctatac tgaaaatttg aacaggaaat atccttttgt
720


 


gtttcctgct gcaaattcaa ggatatagca atgatgagta gattttagtg cgtaattata
780


 


tgttggacga gaatttataa aatcaaggag cagcattcct tgtaaaaaaa aaaaaaaaaa
840


 


aaa
843


<212> Type: DNA


<211> Length: 843


SequenceName: CcCAD1ap partial cDNA


 


Sequence (SEQ ID NO: 8)


<213> OrganismName: Coffea canephora


<400> PreSequenceString:


atcgattacc agaaatgtgt acaaatagac caaaccctta caaaatcttt gtctgttgga
60


 


acaagatctg tgtttgttgg aacaagaatt cacactctgc actcattttt tcacagaaaa
120


 


ggcggcaata atgaagacag tgtgtgtaac aggggcttcg ggctacatag catcatggct
180


 


tgtcaagttc ttgctccagc gtggttacac tgtcaaggca tctgttcgtg acctcaatga
240


 


tccaaagaaa gtagaacact tgcttgcact tgatggagct aaggagagac ttcagttgtt
300


 


caaagcaaac ctactggagg aaggttcctt tgatgctgcg attgatggtt gtgatggtgt
360


 


ttttcatatg gcctctcctt tctaccatac agtcactgat cctcaggctg aattgattga
420


 


tcctgctcta aaagggactc ttagtgttct gggatcatgt gcaaaatctc catctgttaa
480


 


aagagtggtt ttaacatcct ctatagctgc agttgccttc aacggcaagc ctcgtactcc
540


 


ggacgtggtg gttgatgaga cttggtggtc tcttcctgaa ttttgcaagc aaatgaagtt
600


 


atggtatgtt ctctcaaaga cattggctga ggatgctgcc tggaagtttg taaaagagaa
660


 


aggtttagac atggtaacga taaacccagc tatggtgatt gggcctctgt tacagccaac
720


 


acttaacacc agtgctgctg caattttgaa tttaataaac ggtgcagaaa catttccaaa
780


 


ttcttctatg ggctgggttg atgtcaaaga tgttgctaat gcacatattc tagcttttga
840


 


aaatccatct gctagtggaa gatactgcct ggttgaaaga gtggtacact actctgaagt
900


 


tgtgaacatc ttgcgtgaaa tttatccttc ctcaaaactt ccagaaaagt gcgctgatga
960


 


caagccattt gtgccgacgt accaggtttc caaggagaag gcaaaaagct taggtcttga
1020


 


attcattccc gttgagcaaa gcatcaagga aacagttgaa agcttgaagg agaagaactt
1080


 


tttgaactct tctgctgcac tttgaatatc acccgaaaca tgatgaggaa catcgagagt
1140


 


tggtagcatt gcctatttaa aatatgttgt ccaaactcct atatgtacca agcccgtctg
1200


 


actggcgtta catgcatcca cagagattta gaatctacat atgagacaat aaagtactcg
1260


 


tcttttttgc tatactgaaa atttgaacag gaaatatcct tttgtttttc ctgctgcaaa
1320


 


ttcaaggata tagcaatgat gagtagattt tagtgcgtaa ttatatgttg gacgagaatt
1380


 


tataaaatca aggagcacgc atttccttgt aatatcaggg taacgacttt ataaattaaa
1440


 


aaaaaaaaaa aaaaaaa
1457


<212> Type: DNA


<211> Length: 1457


SequenceName: CcCAD1b cDNA


 


Sequence (SEQ ID NO: 9)


<213> OrganismName: Coffea canephora


<400> PreSequenceString:


ccaccaaaca ctttcttctc cacctatcat cggcgcctta attctgccaa tacacaaaac
60


 


tcctccattg tttttcattc catcgacttt tgtcatcatc tacttctctc tcaaccaaga
120


 


aaatccccaa aaaaatactg caaatgtttt agcaatataa cccagcagga gaaaagtcaa
180


 


cacttctgag cccttttaaa ggttacagaa acttaggagc agaggaaggg ggggagccat
240


 


cgcagcttgg cgctgccgtt ttcgccctgc tttcttcagc aaataaccct tgaaaaagag
300


 


acagaatgag cggagcggga aaggtggtgt gcgtgacagg agcatcagga tacatagctt
360


 


cttggttgat caagatgttg cttcatcgtg gttataccgt caaagcttca gttcgtgacc
420


 


tcaatgatcc aaaaaagaca gaatttttga tggcactgga tggagccaag gaaagacttc
480


 


acttgtttca tgcaaactta gtggaagagg gatcctttga tgcaatagtt gatggatgtg
540


 


aaggtgtttt tcatactgca tctccaatcc ttcatccagc gagtgtgacc aatccacaga
600


 


tagagcaatt ggatcctgca ctaaagggaa cactgaatgt gctgcgatct tgtgctagag
660


 


tttcatctat caaaagagtg gttttaacat cttctatgag agcagtgaca tgtaatcggg
720


 


aactaaagga tggtgttgta gttgatgaaa gttggtttgc agatccaaca tactgtgagg
780


 


agcgcaagtt gtggtatcca ctatcaaaga ttttggcaga gaatgctgct tgggaattct
840


 


caaaggagca tggtattgat atggttgcaa tcataccagg aatggtcatt ggtcccatct
900


 


tgcagcctta tcccagttta actgcaggaa tggtcctgaa cgtagtaaaa ggagctgcat
960


 


cgttctatac cgcgcgcatg agatgggttg atgttagaga tgttgcatat gcacatattc
1020


 


tagcctttga agtcccttct gccagtggaa gatattgtgt agttgaggga tttgcactgt
1080


 


ggactgagtt tatcaagact ctgaatgaat tgtatcctac tctccaactg tcagatgagt
1140


 


gttctactag tactccccta gttgagccac actacgaatt atcaaatgag aaagcaaagg
1200


 


gtttggggat tgagttcatt cctttcgacg tttgcctcaa ggataccatc gaaagcttca
1260


 


aagagaagaa cttggttaac ttctgaacaa aattcctaag cagactgaga ttggaaaata
1320


 


tacagtatcc aactattcat tctcttgtgt gagagtaaac tcttaaaagt atgggactgg
1380


 


tgaactttta ttgtagttga taattgtctc tggtactttt attgaactga tcgaacttta
1440


 


attgtagcaa tatgaaacca tgggagggct atgccaagga aggcttaatc caaaaaaaaa
1500


 


aaaaaaaaaa aaaaaaaaaa a
1521


<212> Type: DNA


<211> Length: 1521


SequenceName: CcCAD2 cDNA


 


Sequence (SEQ ID NO: 10)


<213> OrganismName: Coffea canephora


<400> PreSequenceString:


ctttaagcag tggtaacaac gcagagtacg cgggggagtc tgccagatgg atggatactc
60


 


tcatatccat tctgctacta atggccccgt aaggtgatcc ttggatgcaa ggctgacagg
120


 


taacaaattc gctcacaaaa agagcttgga gaagaatgag tggaccagga gaggataaag
180


 


tggtgtgtgt gactggagct tcgggttaca tagcttcatg gctggtcaag cagctacttg
240


 


gccggggtta tacagttaaa gcttctgttc gagatgccaa tgatccaaga aagacggaac
300


 


atttgacgtc acttgatgga gccaaggaga gactgaagtt gtttcaggcg aacttacttg
360


 


atgatggatc ctttgatgaa atagttcaag gatgtactgg cgtttttcat accgcttctc
420


 


ctgttaattt ttcggttagc gatccgaaga aagaattgct agaccctgca gtaaagggaa
480


 


cactgaacct gcttcaatca tgtgcaaaag tttcatctat cagaagagta atcctgacat
540


 


cttctacggc tgcagttctg gcaaaaccag agctaaataa agattcattt gttgacgaaa
600


 


gttggttttc taacccatca tactgtgagg agcaaaagat gtggtatcaa ctgtcaaaaa
660


 


ctttagcaga ggatgctgct tggaaattct caaaggagca tggcattgac atggtttcaa
720


 


tcaatccagg atgggtcttt ggtcccattt tgcagccttc tatcaatcta agtgcaggat
780


 


tggtcctgga tgtagtaaat gggtctcaat catttcctga tgcatgtgtt ggatggattg
840


 


atgttagaga tgttgcctgt gcacatattc atgcctttga aatcccttct gctaatggaa
900


 


gatattgtgt agttgggaaa aatgtgcact ggtccgagat cgtcaagatt ctgaggcaac
960


 


ttttccctac tcttcaactt ccaaataaag gttctcctaa tagcaccttc ggtatgggag
1020


 


aattcgaagt gtcaatggag aaaacaaaag gtttgggaat caatttcatt ccattggaag
1080


 


tgagcctgaa ggacactgtt gaaagtttca tggagaagaa ttttattact ttttaaatca
1140


 


agaacggttt aaatttgtaa gaatttgtgt tcctaaaaca atcataaaga ttatttgtta
1200


 


tagttcatgg agttgtataa ttaaaaaaat tagtcaatgt tcgtaccatc tgttgtatct
1260


 


attgtaatta ttgattcttg gaaaaaaaaa aaaaaaaaaa aaaaaaaaa
1309


<212> Type: DNA


<211> Length: 1309


SequenceName: CcCAD3 cDNA


 


Sequence (SEQ ID NO: 11)


<213> OrganismName: Coffea canephora


<400> PreSequenceString:


gctgaatgtg ctacagtctt gtgccaaagt tcaatctatc aaaagagtga ttttaacgtc
60


 


ttctattgca gcagttatat acaaagatga attaaaggac ggtgtcatag ttgacgaaag
120


 


ttggttttca gttccattat actgtgaaga gcacaagtta tggtatcaac tatcaaaaat
180


 


tttggcagag aatgctgctt gggatttctc aaaggagcat ggtattgaca tgattgcaat
240


 


taatccagga atggtcaccg gtcccttctt gcagccttct gccactttga gtgcagaagt
300


 


gatcttgagn cctagtaaat gtggaagata ttgcgtagtt gagagaactg caggctgctg
360


 


tgagcttatc aggattctga ctgaactctt cccaactctc cagttgccag ataaatattc
420


 


taatggcagt cccctaattc agctgaaata tgatgtatca aatgaaaaag taaaaggttt
480


 


gggcattgag ttcatgcctt tggaggtgag cctcaaggat actatcgaaa gcttcataga
540


 


gatgaaatta gttagccttt gaatcatggc attcatgtac tgtcatcaac ttgaaattgt
600


 


ttatatccaa gtttggaaaa cattcaattt caaacttctc attcttgttt gtacaaggta
660


 


cgcgattcag attatatata aagtagctaa attgtaaata aaaaaaaaaa aaaaaaa
717


<212> Type: DNA


<211> Length: 717


SequenceName: CcCAD4p partial cDNA


 


Sequence (SEQ ID NO: 12)


<213> OrganismName: Coffea canephora


<400> PreSequenceString:


gccacacaaa aggtcgttat tcccctctca attccctctc tcggccctcg tctttcccta
60


 


aaacaaaaaa aatttccagc acacttccac tacacaaaca cacaccaata agaaactttc
120


 


cctcaccctc tcggctctat ctctccattt tttcaattca cacaacacac ccacaaacaa
180


 


ttcaaacaca acatagcaag caacaaagga ttaaagcttg ggagttcctt caaccatttg
240


 


gccgagaaac tcctacttgg attgaggaaa aagagacaga atgagcggag cgggaaaggt
300


 


ggtgtgcgtg acaggagcat caggatacat agcttcttgg ttgatcaaga tgttgcttca
360


 


tcgtggttat accgtcaaag cttcagttcg tgacctcaat gatccaaaaa agacagaatt
420


 


tttgatggca ctggatggag ccaaggaaag acttcacttg tttcatgcaa acttattgga
480


 


agatggatcc tttgatgcaa tagttgatgg atgtgaaggt gtttttcata ctgcatctaa
540


 


caatccttca tccagcgagt gtgaccaatc gaaatagagc aattgcatcc tgcccaaaag
600


 


gaaaaactga aatgtccgtt atctagagca agagctaatt caaatcatcg gataggattt
660


 


aaaatactac aaagaaaaaa tcgcgcttgt aattcgaaac taaataaagc tgttggaagt
720


 


gcaccaaaac tccttataaa tcaa
744


<212> Type: DNA


<211> Length: 744


SequenceName: CcCAD5p partial cDNA


 


Sequence (SEQ ID NO: 13)


<213> OrganismName: Coffea canephora


<400> PreSequenceString:


aaactcacac atttacaatc aatcacagag agaatgagcg gagcagggaa ggtggttggt
60


 


gtgacgggag cttcagggta cgtagcttca tggctggtga agctgctgct tgagcggggt
120


 


tacactgtta aagcttccgt tcgtgacctc aatgatccag acaacacaga acatttgatt
180


 


tcacttgatg gagccaagga aaggcttcac ttgtttgttg ctgacttgat gaaagatgga
240


 


tcatttgatg aaatggttga tggatgtgaa ggtgtttttc atactgcatc tccattcaaa
300


 


cctgtagtta gtgatccaga ggcagaattg ttggaccctg cggtgaaggg aactctgaat
360


 


gtgctacaat catgtgcaag agtttcatct gtcaaaagag tggtagtgac atcttctata
420


 


gcctcggttg catacaaccg agaagcgaag gatggtgttg tagttgatga aagttggttt
480


 


tcagagccat catactgtga agaacgcaag ctctggtatg tactttcaaa aacgttggca
540


 


gagactgctg catggaagtt ctcaaaggag catggcattg atatgattac aattcatcca
600


 


tcatggatca ttggtcctca tttgcagcct tctatcaata caagtgtgca attgatcctg
660


 


aacctactaa atggggatga atcatttcct tacgcaa
697


<212> Type: DNA


<211> Length: 697


SequenceName: CcCAD6p partial cDNA


 


Sequence (SEQ ID NO: 14)


<213> OrganismName: Coffea canephora


<400> PreSequenceString:


tttgaatttg atcgccgcca catcaaagca gttttggtgg acttgcttac tgcagcaatg
60


 


gacactacag ctacaactgt tgaatggata ctcgcggagc tcttgaaaaa cccccgagta
120


 


atgaagaaag tccagcaaga attggacgaa aaagtaggcc tacacaggat ggttgaggaa
180


 


tcagaattgg aaaacctgac atacttagac atggttgtaa aggaagcatt aaggctccat
240


 


cctgttgtac cattacttct tcctcatgca gccttggagg attgcatagt tgacggtttc
300


 


catataccga aagattcccg agtgacgatc aatgcttgga cgatcggaag agatccaaat
360


 


gcatggtctg atcctgagaa gtttacacca gagagattta ttgggagcaa catagatgta
420


 


agaggacatg atttccagct tattcccttt ggctcaggca gaagaatctg ccctggaatg
480


 


cagttggggc taactgtggt ccgtcttatg ttggcacgaa tggtgcattg tttcaattgg
540


 


gaacttccaa atgggatgct gccttcagag ctagacatga ccgaggaatt tggccttgtg
600


 


atgaccaggg ccaagcatct gatggctatt ccaacatatc gattgagcaa atgattaagc
660


 


aggtcttgag agatgcatgc tatttctata ttgcacctta tatattaagg tctactytgc
720


 


taagttcttg aaatgataaa tggttgtcaa gaataggtag gtattcatga cagaaccaag
780


 


tacttttaaa agcttcttct tttgtcactt ataaagtatg aaagatggaa tctttttcac
840


 


aaaattctgt acagtttaaa taaggattac tgcattttty ttttatattc tcttataaag
900


 


tatgcatgtt aatttgaaaa aaaaaaaaaa aaaa
934


<212> Type: DNA


<211> Length: 934


SequenceName: CcF5Hp partial cDNA


 


Sequence (SEQ ID NO: 15)


<213> OrganismName: Coffea canephora


<400> PreSequenceString:


MAEEEACLFA MSLASASVLP MVLKSAIELD LLELIAKAGP GAYVSPSELA AQLPTHNPEA
60


 


PIMLDRILRL LATYSVLDCK LNNLADGGVE RLYGLAPVCK FLTKNADGVS MAPLLLMNQD
120


 


KVLMESWYHL KDAVLDGGIP FNKAYGMTAF EYHGTDPRFN KVFNQGMSNH STITMKKILE
180


 


VYRGFEGLKT VVDVGGGTGA TLNMIISKYP TIKGINFELP HVVEDAPSHS GVEHVGGDMF
240


 


VSVPKGDAIF MKWICHDWSD DHCRKLLKNC YQALPDNGKV ILAECVLPEA PDTSLATQNV
300


 


VHVDVVMLAH NPGGKERTEK EFEALAKGAG FKEFRKVCSA VNTWIMELCK
350


<212> Type: PRT


<211> Length: 350


SequenceName: CcCOMT1 protein


 


Sequence (SEQ ID NO: 16)


<213> OrganismName: Coffea canephora


<400> PreSequenceString:


YELGNSLLEG GIPFNRVHGM HAFDYPSRDP RYNELFNKGM VGPTAITMKK LLQQYKGFEH
60


 


LQTLVDVGGG LGITLHKIIS KYPSIRGINF DLPHVIENAP SYLGVEHIGG DMFESVPGGD
120


 


AIFMKMILHD WSDDHCLKLL KNCFKALPDH GKVIVVDLVL FVKPDTSAFV KGIFQTDALM
180


 


MTQNPGGKER SESDVRALAI RAGFKDIKLE CCVGSLGVLE LYK
223


<212> Type: PRT


<211> Length: 223


SequenceName: CcCOMT2p partial protein


 


Sequence (SEQ ID NO: 17)


<213> OrganismName: Coffea canephora


<400> PreSequenceString:


MDSSSRATDN VVVEAGLDEQ EEQHFSYAMQ LVTSVSLPMV LLAAIRLDVL EVIAQAGPGA
60


 


QLSPWDIAAQ VGPKNPDAAA MLDRMLQLLA SYSVLTCSVA EAD
103


<212> Type: PRT


<211> Length: 103


SequenceName: CcCOMT3p partial protein


 


Sequence (SEQ ID NO: 18)


<213> OrganismName: Coffea canephora


<400> PreSequenceString:


KFFVQNKTKG GGSLGSVLGL LQDKVFIDSW YQLEDAVRKG GDPFHRAHGT HAFEFLGSDP
60


 


RFNEVFNKAM IHHTAIVINR MLERYKGFEH LKTLVDVGGG LGMNLNIITT KYPSLKGINF
120


 


DLPHVIQHAP AYPGVEHVGG DMFESVPQGD AIFMKWILHD WDDGHCLKLL KNCYKALPDN
180


 


GKVIAVDAIL PVVPDDSARD KATCQADLVV VTQYRGGIER YETEFLALAT AAGFKGISVK
240


 


CFVCNLWVME FYK
253


<212> Type: PRT


<211> Length: 253


SequenceName: CcCOMT4p partial protein


 


Sequence (SEQ ID NO: 19)


<213> OrganismName: Coffea canephora


<400> PreSequenceString:


MAAWEAEKAR TVCVTGAGGY LGSWLVKLLL SRHYTVHATL RNPEDEKYVH LKKLDKAAEN
60


 


LKLFKADLLD YNSISAAIRG CDGVFHVASP VPSGSVPNPE VELVEPAVKG TLNVLKACSE
120


 


ANVKRVVAVS SVAAVVVSPN RHKGEIIDET CWSDGEYCKT TNNWYCYSKT VAESEALQYA
180


 


KETGLDVLTV CPSFVLGPML QHDVNASSLA LIKLLKEGYE EIENKFRDMV DVRDVAEALL
240


 


LVYGRPEAEG RYICSSHLTT TKDTVEILRK NYPNYKYPKR FIEVKDDQGR GNVSSEKLQR
300


 


LGWRYRPVEE TLVDSVESYQ QAGILD
326


<212> Type: PRT


<211> Length: 326


SequenceName: CcCCR1 protein


 


Sequence (SEQ ID NO: 20)


<213> OrganismName: Coffea canephora


<400> PreSequenceString:


MPSVSGQVVC VTGAGGYIAS WIVKLLLEKG YTVRGTVRNP DDAKNGHLRE LEGAKERLTL
60


 


CRADLLDYQS LREAIKGCDG VFHTASPVTD DPEQMVEPAV IGTKNVINAA AEAKVRRMVF
120


 


TSSIGAVYMD PNREPEKVVD ESCWSDLEFC KNTKNWYCYG KAVAEKAAWD EAKEKGVDLV
180


 


VINPVLVLGP LLQPTVNASI LKILKYLTGS AKTYANSVQA YVHVKDVALA HVLIYETPSA
240


 


SGRYLCAESV LHRGEVVEIL AKLFPEYPIP TKCSDETRPR AKAYKFTNQK LKDLGFEFTP
300


 


VKQCLYETVK SLQEKGQIPL PTQNDKPIKI HY
332


<212> Type: PRT


<211> Length: 332


SequenceName: CcCCR2 protein


 


Sequence (SEQ ID NO: 21)


<213> OrganismName: Coffea canephora


<400> PreSequenceString:


QTKLWYVLSK TLAEDAAWKF VKEKGLDMVT INPAMVIGPL LQPTLNTSAA AILNLINGAE
60


 


TFPNSSMGWV DVKDVANAYT IAFENPSASG RYCLVERVVH YSEVVKILRE IYPSSKLPEK
120


 


CADDKPFVPT YQVSKEKAKS LGLEFIPLEQ SIKETVESLK EKNFLDSSAA L
171


<212> Type: PRT


<211> Length: 171


SequenceName: CcCAD1ap partial protein


 


Sequence (SEQ ID NO: 22)


<213> OrganismName: Coffea canephora


<400> PreSequenceString:


MKTVCVTGAS GYIASWLVKF LLQRGYTVKA SVRDLNDPKK VEHLLALDGA KERLQLFKAN
60


 


LLEEGSFDAA IDGCDGVFHM ASPFYHTVTD PQAELIDPAL KGTLSVLGSC AKSPSVKRVV
120


 


LTSSIAAVAF NGKPRTPDVV VDETWWSLPE FCKQMKLWYV LSKTLAEDAA WKFVKEKGLD
180


 


MVTINPAMVI GPLLQPTLNT SAAAILNLIN GAETFPNSSM GWVDVKDVAN AHILAFENPS
240


 


ASGRYCLVER VVHYSEVVNI LREIYPSSKL PEKCADDKPF VPTYQVSKEK AKSLGLEFIP
300


 


VEQSIKETVE SLKEKNFLNS SAAL
324


<212> Type: PRT


<211> Length: 324


SequenceName: CcCAD1b protein


 


Sequence (SEQ ID NO: 23)


<213> OrganismName: Coffea canephora


<400> PreSequenceString:


MSGAGKVVCV TGASGYIASW LIKMLLHRGY TVKASVRDLN DPKKTEFLMA LDGAKERLHL
60


 


FHANLVEEGS FDAIVDGCEG VFHTASPILH PASVTNPQIE QLDPALKGTL NVLRSCARVS
120


 


SIKRVVLTSS MRAVTCNREL KDGVVVDESW FADPTYCEER KLWYPLSKIL AENAAWEFSK
180


 


EHGIDMVAII PGMVIGPILQ PYPSLTAGMV LNVVKGAASF YTARMRWVDV RDVAYAHILA
240


 


FEVPSASGRY CVVEGFALWT EFIKTLNELY PTLQLSDECS TSTPLVEPHY ELSNEKAKGL
300


 


GIEFIPFDVC LKDTIESFKE KNLVNF
326


<212> Type: PRT


<211> Length: 326


SequenceName: CcCAD2 protein


 


Sequence (SEQ ID NO: 24)


<213> OrganismName: Coffea canephora


<400> PreSequenceString:


MSGPGEDKVV CVTGASGYIA SWLVKQLLGR GYTVKASVRD ANDPRKTEHL TSLDGAKERL
60


 


KLFQANLLDD GSFDEIVQGC TGVFHTASPV NFSVSDPKKE LLDPAVKGTL NLLQSCAKVS
120


 


SIRRVILTSS TAAVLAKPEL NKDSFVDESW FSNPSYCEEQ KMWYQLSKTL AEDAAWKFSK
180


 


EHGIDMVSIN PGWVFGPILQ PSINLSAGLV LDVVNGSQSF PDACVGWIDV RDVACAHIHA
240


 


FEIPSANGRY CVVGKNVHWS EIVKILRQLF PTLQLPNKGS PNSTFGMGEF EVSMEKTKGL
300


 


GINFIPLEVS LKDTVESFME KNFITF
326


<212> Type: PRT


<211> Length: 326


SequenceName: CcCAD3 protein


 


Sequence (SEQ ID NO: 25)


<213> OrganismName: Coffea canephora


<400> PreSequenceString:


LNVLQSCAKV QSIKRVILTS SIAAVIYKDE LKDGVIVDES WFSVPLYCEE HKLWYQLSKI
60


 


LAENAAWDFS KEHGIDMIAI NPGMVTGPFL QPSATLSAEV ILPSKCGRYC VVERTAGCCE
120


 


LIRILTELFP TLQLPDKYSN GSPLIQLKYD VSNEKVKGLG IEFMPLEVSL KDTIESFIEM
180


 


KLV
183


<212> Type: PRT


<211> Length: 183


SequenceName: CcCAD4p partial protein


 


Sequence (SEQ ID NO: 26)


<213> OrganismName: Coffea canephora


<400> PreSequenceString:


MSGAGKVVCV TGASGYIASW LIKMLLHRGY TVKASVRDLN DPKKTEFLMA LDGAKERLHL
60


 


FHANLLEDGS FDAIVDGCEG VFHTAS
86


<212> Type: PRT


<211> Length: 86


SequenceName: CcCAD5p partial protein


 


Sequence (SEQ ID NO: 27)


<213> OrganismName: Coffea canephora


<400> PreSequenceString:


MSGAGKVVGV TGASGYVASW LVKLLLERGY TVKASVRDLN DPDNTEHLIS LDGAKERLHL
60


 


FVADLMKDGS FDEMVDGCEG VFHTASPFKP VVSDPEAELL DPAVKGTLNV LQSCARVSSV
120


 


KRVVVTSSIA SVAYNREAKD GVVVDESWFS EPSYCEERKL WYVLSKTLAE TAAWKFSKEH
180


 


GIDMITIHPS WIIGPHLQPS INTSVQLILN LLNGDESFPY A
221


<212> Type: PRT


<211> Length: 221


SequenceName: CcCAD6p partial protein


 


Sequence (SEQ ID NO: 28)


<213> OrganismName: Coffea canephora


<400> PreSequenceString:


FEFDRRHIKA VLVDLLTAAM DTTATTVEWI LAELLKNPRV MKKVQQELDE KVGLHRMVEE
60


 


SELENLTYLD MVVKEALRLH PVVPLLLPHA ALEDCIVDGF HIPKDSRVTI NAWTIGRDPN
120


 


AWSDPEKFTP ERFIGSNIDV RGHDFQLIPF GSGRRICPGM QLGLTVVRLM LARMVHCFNW
180


 


ELPNGMLPSE LDMTEEFGLV MTRAKHLMAI PTYRLSK
217


<212> Type: PRT


<211> Length: 217


SequenceName: CcF5Hp partial protein






The present invention is not limited to the embodiments described and exemplified above, but is capable of variation and modification within the scope of the appended claims.

## Claims

1. A nucleic acid molecule isolated from coffee (Coffea spp.), having a coding sequence that encodes a caffeic acid O-methyltransferase enzyme, wherein the caffeic acid O-methyltransferase enzyme comprises an amino acid sequence at least 90% identical to one or more of SEQ ID NOs: 16-18.

2. The nucleic acid molecule of claim 1, wherein the caffeic acid O-methyltransferase enzyme comprises an amino acid sequence greater than 95% identical to SEQ ID NO:16.

3. The nucleic acid molecule of claim 1, wherein the caffeic acid O-methyltransferase enzyme comprises an amino acid sequence greater than 95% identical to SEQ ID NO:17.

4. The nucleic acid molecule of claim 1, wherein the caffeic acid O-methyltransferase enzyme comprises an amino acid sequence greater than 95% identical to SEQ ID NO:18.

5. The nucleic acid molecule of claim 1, wherein the coding sequence is an open reading frame of a gene, or a mRNA, or a cDNA.

6. The coding sequence of the nucleic acid molecule of claim 1, contained within a vector.

7. The vector of claim 6, which is an expression vector selected from the group of vectors consisting of plasmid, phagemid, cosmid, baculovirus, bacmid, bacterial, yeast and viral vectors.

8. The vector of claim 6, wherein the coding sequence of the nucleic acid molecule is operably linked to a constitutive promoter, or an inducible promoter, or a tissue-specific promoter.

9. The vector of claim 8, wherein the tissue specific promoter is a seed specific promoter.

10. The vector of claim 9, wherein the seed specific promoter is a coffee seed specific promoter.

11. A host cell transformed with the vector of claim 6.

12. The host cell of claim 11, which is a plant cell selected from the group of plants consisting of coffee, tobacco, Arabidopsis, maize, wheat, rice, soybean, barley, rye, oats, sorghum, alfalfa, clover, canola, safflower, sunflower, peanut, cacao, tomatillo, potato, pepper, eggplant, sugar beet, carrot, cucumber, lettuce, pea, aster, begonia, chrysanthemum, delphinium, petunia, zinnia, and turfgrasses.

13. A fertile plant produced from the plant cell of claim 12.

14. A method of modulating flavor or aroma of coffee beans, comprising modulating production or activity of one or more caffeic acid O-methyltransferase enzymes within coffee seeds, wherein the caffeic acid O-methyltransferase enzyme comprises an amino acid sequence at least 90% identical to one or more of SEQ ID NOs: 16-18.

15. The method of claim 14, comprising increasing production or activity of the one or more caffeic acid O-methyltransferase enzymes.

16. The method of claim 14, comprising decreasing production or activity of the one or more caffeic acid O-methyltransferase enzymes.