MX2008004864A - Nucleic acids and proteins associated with galactomannan synthesis in coffee - Google Patents

Abstract

Disclosed herein are nucleic acid molecules isolated from coffee (Coffea spp.) comprising sequences that encode mannan synthase or galactomannan galactosyltransferase. Also disclosed are methods for using these polynucleotides for gene regulation and manipulation of the polysaccharide molecules of coffee plants, to influence extraction characteristics and other features of coffee beans.

Description

NUCLEIC ACIDS AND PROTEINS ASSOCIATED WITH SYNTHESIS OF GALACTOMANAN IN COFFEE FIELD OF THE INVENTION The present invention relates to the field of agricultural biotechnology. More particularly, the invention relates to enzymes from coffee plants that participate in polysaccharide metabolism, including galact-omanan synthesis, and the nucleic acid sequences encoding it.

BACKGROUND OF THE INVENTION Several publications, including patents, published applications and school articles, are cited throughout this specification. The complete contents of each of these publications are incorporated into the present, in their totalities. Citations not fully established within the specification can be found at the end of the specification. A key stage in coffee processing is the toasting of green beans. The roasting stage is usually carried out in the range of 170 ° to 230 ° C for 5 to 15 minutes and is responsible for generating most of the aroma, flavor and color associated with the coffee beverage (Yeretzian, et al. 2005). Depending on the degree of toasting, 12-40% of the polysaccharides can degrade at this stage (Redgwell, et al., 2002). It has been reported that the roasting stage alters the length of many of the complex polysaccharide polymers, which can increase their solubility (Redgwell, et al., 2002). Fragmentation of coffee polysaccharides is thought to favorably affect the organoleptic properties of the beverage such as mouthfeel (Illy and Viani 1995) and foam stability (Nunes et al., 1997). It is also thought that analysis of polysaccharides influences the binding of volatile aroma compounds indirectly because some complex carbohydrate degradation products participate in the formation of roasted melanoidins, a class of poorly defined compounds that constitute up to 20% by weight. % of the roasted grain in dry weight (Charles-Bernard, et al., 2005). The splitting of the polysaccharides induced by roasting can also produce an increase in the amount of solids extracted from the coffee bean, a property of critical importance for the production of soluble coffee. Additionally, the fragmentation / degradation of the carbohydrates in the coffee bean also contributes to the generation of an important group of coffee flavor and aroma molecules through the Maillard reaction associated with coffee roasting (Yeretzian, et al. , 2005). Carbohydrates form a large proportion of the ripe green coffee bean (green bean). Approximately 48-55% of the dry weight in green grains of Arabica (Co ffea a rabi ca) and sturdy (C. ca n eph ora) is composed of carbohydrate, some of which is in the form of complex polysaccharides, while other forms include free mono- and di-saccharides (Clifford MN, 195 in Coffee: Botany, Biochemistry, and Production, pp 374, ed Clifford, M. and Willson, K., Crom Melm Ltd, London, Fischer, et al., 2001, Carbohydrate Research, 330, 93-101). Three major types of carbopleted polymers based on carboplerate have been identified in the coffee bean. The most abundant grain polysaccharides are galactomannans, which are reported to account for up to 25% of the mass in the mature green coffee bean, that is, approximately 50% of the grain carbohydrates. (Oosterveld et al., 2003 Carbohydrate Polymers 52, 285-2960). The next most abundant group of polysaccharides are the arabinogalactans that comprise up to 35% of the green-grained polysaccharides (Oosterveld et al., 2003, s upra). Approximately the remaining 16% of Arabica green grain polysaccharides consists mainly of cellulose and xyloglucans (Oosterveld et al., 2003). Manan containing hemicelluloses is composed of a structure of linked beta 1-4 mannose molecules, and although they can be easily found in mannanose plants they have been considered to be a relatively minor constituent in the walls of most types of plant cell (Bacic, Harris, and Stone 1988, Fry 2004, Somervi'lle, et al., 2004b). Some endosperm containing seeds, such as those of Legu inosae, Palmae, and commercially important coffee species, have very large amounts of galactomannans in the walls of seed endosperm cells (Matheson 1990; Buckeridge, et al. L. , 2000; Pettolino, et al. L. , 2001; Redgwell et al. L. , 2002; Hanford, et al., 2003). Galactomannans are characterized by the chains of mannan that have unique galactosyl molecules linked by a (1-6) alpha bond. Seed endosperm galactomannans appear to be associated with thickening of the secondary cell wall of the endosperm cell wall (Pettolino, et al., 2001; Sunderland, et al., 2004; Somerville et al., 2004a) and it is believed that they are part of the energy reserve of mature seed, which is analogous to the role played by starch in cereal endosperms (Reid 1985). Other functions that have been put into theory for endosperm galactomannans include facilitating the imbibition / germination and protection of the desiccation seed embryo (Reid and Bewley 1979). Other cell wall polymers based on main mannan include glucomannans that have some of the mannose units replaced by beta-1,4-glucose residues-inlays, and galactomannans that are glucomannan residues. galactose alpha-1,6-linked. Galactomannans with low galactose levels are important constituents of thick lignified secondary cell walls of gymnosperms (Lundqvis t, J., Et al., 2002) and have also been found in kiwi fruit (Actinidia deliciosa) and tobacco cells grown in tissue (Nicotiana plumbagini folia) (Schroder, R., et al., 2001; Sims, I., et al., 1997). Recent studies have shown that mannan polymers exist in the thick secondary cell walls of xylem elements, xylem parenchyma, and fasicular inner fibers of the angiosperm model Arabidopsis thaliana (Handford et al., 2003). They also detect significant levels of mannan in the thick epidermal cell walls of leaves and stems, and lower levels of mannans in most of the other tissues examined, indicating the widespread presence of mannan in arabidopsi s. Although cellulose polymers are known to be synthesized in the plasma membrane, most non-cellulosic polysaccharides are thought to be made in the golgi apparatus and then transported out of the cell membrane into the apoplastic space (Keegstra and Raikhel 2001). Somerville, Bauer, Brininstool, Facette, Hamann, Milne, Osborne, Paredez, Persson, Raab, Vorwerk, and Youngs 2005, Liepman, Wilkerson, and Keegstra 2005b). Two membrane-bound glycosyltransferases are known to be included in the synthesis of galactomannans: one dependent on Mg + +, (1, 4) -beta-D-mannosyl transferase or mannose synthase (MS) dependent on GDP-Man and one dependent on Mn ++, (1, 6) -alpha- D-galactant os t rans ferase (GMGT) dependent on UDP-Gal, and these enzymes are believed to work together very closely to determine the statistical distribution of galactosyl residues as long of the manan polymer (Edwards, Choo, Dickson, Scout, Gridley, and Reid 2004). Confirmation that mannans are synthesized in the golgi apparatus has been recently obtained by using mannan-specific antibodies to detect mannan in vi t ro synthesis, and this also supports the overall model in which hemicellulose-like polysaccharides such how galactomannans are made in golgi and then transported to the cell membrane and secreted in the apoplast region (Handford, Baldwin, Goubet, Prime, Miles, Yu, and Dupree 2003, Somerville, Bauer, Brininstool, Facette, Hamann, Milne , Osborne, Paredez, Persson, Raab, Vorwerk, and Youngs 2005). The importance of a golgi-linked GMGT protein in the synthesis of seed endosperm galactomannans, and more precisely to control the level of galactose modification, has recently been demonstrated by showing that either the over-, or sub-expression of the GMGT protein from Lotus japonicus causes predictable changes in the ratios of galactose / randosa in the seed (Edwards, Choo, Dickson, Scout, Gridley, and Reid 2004). Until recently, the genes responsible for the synthesis of plant cell mannan were not known. The first isolated gene coding for a biochemically demonstrated mannan synthase was Mansa from seeds of Cyamopsi s t e t ra gonol oba (guar) (Dhugga, et al., 2004). The CtMans cDNA is isolated from EST libraries made from three different stages of development of guar seed, a seed that makes very large amounts of galactomannans. ESTs related to CtManS are identified when looking for sequences with strong similarities with plant CelA (cellulose synthases generating beta-1,3-glucans) and Csl (cellulose synthase-like proteins). The Csl genes have significant similarity to the CelA genes, and have been previously proposed as' candidate genes for enzymes included in the synthesis of hemicellulose as galactomannans (Cutler and Somerville 1997; Richmond and Somerville 2000; Hazen et al. , 2002X The abundance of maize candidate synthase ESTs in each guar seed library corresponds to the mannan synthase activity levels biochemically measured at each stage, suggesting that these ESTs represent a manan synthase. The putative mannan synthase cDNA is shown to encode a functional enzyme by showing that somatic embryos from soybeans, which normally do not have detectable mannase synthase activity, showed significant mannan synthase activity when they over-express the sequence of CtMan's cDNA (Dhugga et al., 2004). It is found that the recombinant functional enzyme is located in the golgi apparatus. In the arabidopsis genome, there are more than 25 genes annotated as Csl genes and these are subdivided into families based on their sequence homologs. Recently, a functional evaluation has been carried out on recombinant proteins generated from a number of the sequences of the Csl gene of arabidopsis and it is determined that several members of the CslA gene family encoded proteins with beta-mannan synthase activity (Liepman , etal., 2005). There is little information available on the metabolism of polymers related to mannan in coffee. Several highly related cDNAs that encode a fa-galactose or idasa found in coffee bean have been obtained and the expression of this gene to develop grain indicates that this gene is induced during the formation and expansion of the endosperm (approximately 22-27 WAF ( Weeks after Fertilization) and expression can also be detected in leaves, flowers, zygotic embryos, and weakly in roots (Marraccini et al., 2005) .The galactose / mannose ratio of the galactomannans in the coffee bean falls from a ratio of approximately 1: 2 to 1: 7 at an early stage of grain development (11 WAF; weeks after fertilization) at a ratio of 1: 7 to 1:40 near maturity at 31 WAF (Redgwell, et al. , 2003) This information, together with the expression data under development for alpha-galactosidase presented above, leads to the theory that this particular alpha-galactosidase gene product could be directly included in the decrease in galactose content of coffee bean galactomannans beginning around 21-26 WAF and continuing until grain maturity (Redgwell, et al. , 2003). Support for this model is found in the development of sena seeds (Western Senna) where it was found that a significant increase in alpha-galactosidase activity coincides with the reduction of the galactose content of seed galactomannans (Edwards, et al., 1992). Additional support for the inclusion of an alpha-galactosidase in the reduction of the galactose content is subsequently obtained when sena alpha-galactosidase is expressed to develop seeds of Cyamopsis tet ragonoloba (guar) with the help of a seed-specific promoter (Joersbo, et al., 2001). Guar seeds have high galactomannan levels that have a very high proportion of galactose / mannan, but guar seeds produced from plants expressing sena at fa-galact osidase showed significant reductions in the level of galactose / mannose ratio in modified guar seeds . Two cDNAs encoding different endo-beta mannanases (manA and manB) have also been isolated from the germinating coffee bean (Marraccini, et al., 2001). The corresponding genes are not expressed in the developing grain, but both are expressed during germination, with transcripts detected beginning 10-15 days after imbibition. This observation suggests that both of these manases are associated with the degradation of galactomannans during germination and result in the release of free sugars which then act both as a source of energy and reduced carbon for the germinating seed. The expression of manA is examined and no expression is detected in leaves, somatic embryos, flower buds and roots (Marraccini, et al., 2001). Despite the abundance of galactomannans in coffee beans and the implicit importance of enzymes involved in. synthesis of galactomannans, little information is available in these genes in coffee. Thus, there is a need to identify, isolate and characterize the enzymes, genes, and genetic regulatory elements included in the biosynthetic pathway of galactomannan in coffee. Such information will allow the synthesis of galactomannan to be genetically engineered, with the aim of imparting desirable phenotypic benefits associated with the production of altered galatomannan.

BRIEF DESCRIPTION OF THE INVENTION One aspect of the invention comprises a nucleic acid molecule isolated from Coffea spp. which comprises a coding sequence encoding a galactomannan synthesis enzyme, which may be a galactose i 1 trans ferase or a mannan synthase. In certain embodiments, the mannan synthase comprises a conserved domain having the amino acid sequence QHRWS. In other embodiments, the mannan synthase comprises an amino acid sequence greater than about 75% identical to that of any of SEQ ID NOS: 4-6 ,. and preferably they comprise any of SEQ ID NOS: 4-6. Specifically, the coding sequence comprises SEQ ID NO: 2 or SEQ ID NOX. In other embodiments, the enzyme is a galactyl transferase having at least about 54% amino acid sequence identity with a galactosyl ransferase of fenugreek or a galactyl osyl trans ferase from Lo t u s j aponi cus. In other embodiments, the galactose trans transferase comprises an amino acid sequence greater than about 75% identical to any of SEQ ID NOS: 15-18, and preferably comprises any of SEQ ID NOS: 15-18. Specifically, the coding sequence comprises any of SEQ ID NOS: 11-14. In certain embodiments, the coding sequence is an open reading frame of a gene, or a mRNA molecule produced by transcription of a gene, or a cDNA molecule produced by reverse transcription of the mRNA molecule. Another aspect of the invention comprises 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 comprises a vector comprising the coding sequence of the nucleic acid molecule described above. The vector can be an expression vector selected from plasmid, phagemid, cosmid, baculovirus, bacmid, bacterial, yeast and viral vectors. In certain embodiments, the coding sequence of the nucleic acid molecule is operably linked to a constitutive promoter. Alternatively, it is operably linked to an inducible promoter. In another alternative, the coding sequence of the nucleic acid molecule is operably linked to a tissue-specific promoter, which can be a specific promoter of seed in certain modalities, and more particularly a specific promoter of coffee seed. Another aspect of the invention comprises a host cell transformed with the aforementioned vector. The host cell can be selected from plant cells, bacterial cells, fungal cells, insect cells and mammalian cells. A fertile plant produced from a transformed plant cell is also provided. Another aspect of the invention comprises a method for modulating the extraction of solids from coffee beans, which comprises modulating the production or activity of galactomannan synthesis enzyme with coffee seeds. Specifically, the enzymes are galactose i 1 trans ferase or mannan synthase, or a combination thereof. In one embodiment, the production or activity of the galactomannan synthesis enzyme is increased, for example, by increasing the expression of a gene encoding the enzyme, or by introducing a transgene encoding the enzyme. In another embodiment, the production or activity of the galactomannan synthesis enzyme is decreased, for example, by interfering with the expression of a gene encoding the enzyme. Other features and advantages of the invention will be understood by reference to figures 1 to 12, detailed description and exemplary examples.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Illustration of the galactomannan polymer structure. Figures 2A and 2B. Isolation and characterization of the complete coding sequences for mannan synthases that encode Coffea canephora and Coffea arabica nucleic acids. Figure 2A. Overview of the clones used to identify the complete ORF sequence for CcManS encoding the mannan synthase of C. canephora and CaManS of mannan synthase encoding C. arabica. Four partial cDNA clones are obtained, which cover complete CcManS ORF (see Examples): two RAZA 5 'products, pVC2 and pVC3 containing the 5' end coding sequence of CcManS, and two partial cDNA clones (fish 46w24 cl 9 and pcccs46wl6il 1), containing the remaining 3 'end of CcManS. The cDNA clones pVC4, pVC6 and pVC7 contain PCR amplified sequences containing the complete open reading frames encoding the coffee mannan synthase (Note: pVC4 contains a stop codon at 1118 bp due to an error introduced during "the stage PCR amplification, as discussed in the examples.) The observations are as follows: fish 6wl 6 and 11 = cDNA clone insertion sequences cccs46wl6ill (with two introns and 3 'non-coding sequences in the removed clone ) of Coffea canephora (SEQ ID NO: 7), pcccs 6w2 cl 9 = cDNA clone insertion sequences cccs46w24cl9 of Coffea canephora (SEQ ID NO: 8), pVC2 (SEQ ID NO: 9) = first RAZA fragment Coffea canephora , Robusta var (BP409), cloned in pCR-4-Mole, pVC3 (SEQ ID NO: 10) = second RACE fragment, cloned in pCR-4-Mole; pVC4 (SEQ ID NO: l) = full length amplification of mannan synthase encoding Coffea canephora polynucleotide, Robusta var. (BP409), cl ontop in pCR-4-Mole (this fragment has a stop codon in ORF); pVC6 (SEQ ID NO: 2) full-length amplification of CcManS, a polynucleotide encoding mannan synthase from Coffea canephora, var. Robusta (BP409), cloned in pCR-4-Topo; pVC7 (SEQ ID NO: 3) = full length amplification of CaManS, a polynucleotide encoding mannan synthase of Coffea arabica, var. Arabica (T2308), cloned in pCR-4-Topo. Figure 2B. Alignment of all sequences for CcManS (SEQ ID NO: l and SEQ ID NO: 2) and CaManS (SEQ ID NO: 3) performed using the CLUSTALW program (Lasergene package, DNASTAR) and manually optimized. The nucleotide in a circle in the pVC4 sequence marked the mutated base leading to the stop codon in the ORF of this clone. However, it is clear that the other three cDNA sequences encoding this region, all of which are from independent PCR reactions, have an A instead of a T in this position leading to the expected protein. Therefore, we believe that this T in pVC4 is due to PCR or abnormality in cloning. The gray sequences are coupled with pVC6. The intron sequences are observed by the presence of a black line above this sequence. A deletion in the pVC3 sequence at position 325 induces a change in the open reading frame and is believed to be an error generated during the RT-PCR cloning of this sequence. Figure 3. Shows the complete protein sequence of CcManS from Coffea ca n eph ora (SEQ ID NO: 5). This protein sequence is deduced from the cDNA sequence encoded by pVC6 (SEQ ID NO: 2). Figure 4. Alignment of the protein sequence of the coffee mannan synthase sequences with other mannan synthase sequences. The CcManS protein sequences (SEQ ID NO: 5) deduced from the pVC4 and pVC6 sequences and the CaManS protein sequence (SEQ ID NO: 6) deduced from the pVC7 sequence are aligned with other available mannan synthase proteins at the NCBI database using CLUSTALW, followed by an optimization stage m. annual (Note: the stop codon in pVC4 at position 345 is marked by a red circle). The regions reported to be conserved in ß-gl icosyl trans ferases are either marked by a * or are framed (as in Dhugga et al., 2004). The amino acids marked in the gray combination represent the amino acids most frequently found in that position. The access numbers for the sequences used are CtManS biochemically characterized (Cyamops i s tetragonoloba, AAR23313, SEQ ID NO: 21), AtManS (Arabidpsis thaliana, CAB82941, SEQ ID NO: 22), and IbManS (Ipomoea trífida, AAQ62572; SEQ ID NO: 23). Figure 5. Shows the sequence alignment of the protein sequence (SEQ ID NO: 15) of unigen 122620 (SEQ ID NO: 11) with two biochemically characterized plant GMGT sequences. The partial ORF of unigen 122620 (CcGMGTl) is aligned with the protein sequences of GMGT Lotus j aponicus (accession number AJ567668, SEQ ID NO: 24) and GMGT fenugreek (Trigonella foenum-graecum) (access number AJ245478, SEQ ID NO: 25; observed to be a partial cDNA) using ClustalW. The amino acids found in two or more sequences are in gray. Figure 6. Shows the sequence alignment of the protein sequence (SEQ ID NO: 16) of unigen 122567 (SEQ ID NO: 12) with two GMGT plant sequences biochemically characterized. The partial ORF of unitar 122567 (CcGMGT2) is aligned with GMGT protein sequences Lotus japonicus (accession number AJ567668, SEQ ID NO: 24) and GMGT fenugreek (Trigonella foenum-graecum) (accession number AJ245478, SEQ ID NO. : 25); observed to be a partial cDNA) using ClustalW. The amino acids found in two or more sequences are in gray. Figure 7. Schematic representation of the three clones encoding complete or partial ORF sequence data for coffee GMGTase 1. pcccs46w8o23 (SEQ ID NO: 19) is an EST library clone of C. canephora, pVClO (SEQ ID NO: 20) contains the RAZA 5 'sequence isolated, and pVCll (SEQ ID NO: 13) contains the Arabian genomic fragment that contains the complete polypeptide sequence of GMGTase 1 arabica. Figure 8. Alignment of the DNA sequences GMGTase 1 of pcccs46w8o23 (SEQ ID N0: 19), pVClO (SEQ ID NO: 20), and pVCll (SEQ ID N0: 13) with the generated sequence "in silico" of unigen # 122620 (SEQ ID NO: 11). The alignment is done using CLUSTALW and adjusted manually. Figure 9. Alignment of the protein sequence of CaGMGTase 1 (SEQ ID NO: 17) with the most homologous protein sequences found in the GenBank public database. The alignment is done using CLUSTALW. Accession numbers: CAB522 6: [Tri gonel la foenum-graecum] Alpha galactosyltransferase (SEQ ID NO: 26); CAI11452: [Solanum tuberosum] Alpha-6-galactosyltransferase (SEQ ID NO: 27); CAI11453:: [Nicotiana ben thamiana] Alpha-6-galactosyltransferase (SEQ ID NO: 28); CAI11454: [Medicago trunca tula] Alpha-6-galactosyltransferase (SEQ ID NO: 29); ABE79594: [Medicago trunca tula] Gal actsil transiera se (SEQ ID NO: 30); CAI79 02: [Cyamopsis tetragonoloba] Galactomannan galactosyltransferase (SEQ ID NO: 31); CAI79403: [Senna occidentalis] Galactomannan galactosyltransferase (SEQ ID NO: 32); CAD9892: [Lo tu corniculatus var. j aponicus] Galactomannan galactosyltransferase (SEQ ID NO: 33). Figure 10. Alignment of the DNA sequences GMGTase 2 of Unigen # 122567 (SEQ ID NO: 12 with the DNA sequence of C. canephora GMGTase 2 cDNA clone pcccl26f9 (CcGMGTase 2; SEQ ID NO: 14) using CLUSTALW. 11. Alignment of the protein sequence of CcGMGTase 2 (SEQ ID NO: 18) with CaGMGTase 1 (SEQ ID NO: 17) or the most homologous protein sequences found in the GenBank public database.The alignment is done using CLUSTALW Accession numbers: CAB522 6: [Trigonella foen um-graecum] Alpha galactosyltransferase (SEQ ID NO: 26); CAI11452: [Solanum t ueerosum] Alpha-6-galactosyltransferase (SEQ ID NO: 27); CAI11453: [Nicotiana ben thamiana] Alpha-6-galactosyltransferase (SEQ ID NO: 28); CAI11454: [Medicago trunca tula] Alpha-6-galactosyltransferase (SEQ ID NO: 29); ABE79594: [Medicago trunca tula] Galactosyltransferase (SEQ ID NO: 30 ); CAI 79402: [Cyamopsis tetragonoloba] Galactomannan galactosyltransferase (SEQ ID N0: 31); CAÍ 794 O 3: [Senna occidentalis] Galactomannan galactosyltransferase (SEQ ID NO: 32); CAD98924: [Lo t u s corn i c u l a t u s va t. j apon i c u s] Galactomannan galactosyltransferase (SEQ ID NO: 33). Figure 12. Quantitative RT-PCR expression for CaGMGTl in several tissues of Coffea ca n eph ora and Coffea Arábi ca. DETAILED DESCRIPTION OF ILLUSTRATIVE MODALITIES Definitions: Various terms that relate to biological molecules and other aspects of the present invention are used throughout the specification and claims. The terms are presumed to have their usual meaning in the field of molecular biology and biochemistry unless they are specifically defined otherwise in the present. "Isolated" meaning 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 an animal or living plant is not "isolated", but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is used at the moment. "Polynucleotide", also referred to as "nucleic acid molecule", generally refers to any ibonucleotide or polynucleotide polideoxir, which may be unmodified DNA or RNA or modified DNA or RNA. "Polynucleotides" include, without limitation, single or double stranded DNA, DNA that is a mixture of single or double filament regions, single or double filament RNA, and RNA which is a mixture of single or double filament regions, hybrid molecules that they comprise DNA or RNA which may be single filament or, more typically, double filament or a mixture of single or double filament regions. In addition, "polynucleotide" refers to triple strand regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes ADSs or RNAs that contain one or more modified bases and DNAs or RNAs with structures modified for stability or for other reasons. Bases. "modified" include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, "polynucleotide" comprises chemically, enzymatically, or chemically modified meth- ods of polynucleotides as typically found in nature, as well as the chemical forms of DNA or RNA characteristic of viruses and cells. "Polynucleotide" also comprises relatively short polynucleotides, often referred to as oligonucleotides. "Polypeptide" refers to any peptide or protein comprising two or more amino acids joined together by peptide bonds or modified peptide bonds, i.e., isoesters of peptide. "Polypeptide" refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. The polypeptides may contain amino acids other than 20 amino acids encoded by gene. "Polypeptides" include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques that 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 search literature. Modifications can occur anywhere in a polypeptide, including the peptide structure, the amino acid side chains and the amino or carboxyl terms. 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 can contain many types of modifications. The polypeptides can be branched as a result of ubiquitination, and can be cyclic, with or without branching. The cyclic, branched and branched cyclic polypeptides can result from natural post-translational processes or can be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosion, amidation, covalent binding 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 phospholipids, degradation, cyclization, formation of disulfide bond, formation of covalent degradations, formation of cysteine, amorphous pyroglyph formation, formylation, gamma-carboxylation, glycosylation, GPI binding formation, hydroxylation, iodination, methylation, miri stoi lation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoi tion, sulfation, RNA-mediated addition of amino acid transfer to proteins such as arginylation, and ubiquitination. See for example, Proteins-Structure and Molecular Properties, 2nd Ed., TE Creighton, WH Freeman and Company, New York, 1993 and Wold, F., Pos tt rans lat ional Protein Modi f ica t ions: Perspectives and Prospects, pgs . 1-12 in Pos tt rans lat iona 1 Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York, 1983; Seifter et al., "Analysis for Protein Modi ficat ions 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: 8-62. "Variant" as the term is used in the present, .is a poly n_uc 1 e_ótido. or polypeptide that differs from a reference polynucleotide or polypeptide, respectively, but maintains 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. The nucleotide changes can result in substitutions, additions, deletions, fusions and truncations of amino acids 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. Usually, the differences are limited so that the sequences of the reference polypeptide and the variant are closely similar in total 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 encoded by the genetic code. A variant of a polynucleotide or polypeptide can occur naturally, such as an allelic variant, or it can be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides can be made by mutagenesis techniques or by direct synthesis. In reference to mutant plants, the terms "null mutant" or "loss of mutant function" are used to designate an organism or genomic DNA sequence with a mutation that causes a genetic 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 nucleic acid regions. These mutations can also occur in the coding and / or regulatory regions of other genes that can regulate or control an encoded gene and / or protein, to cause the protein to be non-functional or largely absent. The term "substantially the same" refers to nucleic acid or amino acid sequences that have sequence variations that do not materially affect the nature of the protein (i.e., structure, stability characteristics, substrate specificity and / or activity). biological 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 that govern expression, and is primarily concerned with degenerating codons encoding the same amino acid, or alternating codons encoding the substituted amino acids conserved in the encoded polypeptide. With reference to amino acid sequences, the term "substantially the same" generally refers to conservative substitutions and / or variations in polypeptide regions not included in the determination of structure or function. The terms "identical percent" or "similar percent" are also used herein "in comparisons between amino acid sequences and nucleic acids." When referring to amino acid sequences, "identity" or "identical percent" refers to percent of the amino acids in the subject amino acid sequence that have been coupled to identical amino acids in the amino acid sequence compared by a sequence analysis program. "Percent percent" refers to the percent of the amino acids in the sequence of subject amino acids that have been coupled to identical or conserved amino acids Conserved amino acids are those that differ in structure but are similar in physical properties so that the exchange of one by the other would not appreciably change the tertiary structure of the resulting protein. Conservative substitutions are defined in Taylor (1986, J. Theor, Biol. 119: 205). Nucleic acid cells, "identical percent" refers to the percent nucleotides of the subject nucleic acid sequence that have been coupled to identical nucleotides by a sequence analysis program. "Identity" and "similarity" can be easily calculated by known methods. The nucleic acid sequences and amino acid sequences can be compared using computer programs that align the similar amino acid or nucleic sequences and thus define the differences In preferred methodologies, the BLAST programs (NCBI) and parameters used in the present they are employed, and the DNAstar system (Madison, Wl) is used to align the 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 example, GCG Wisconsin Package version 9.1, available from Genetics Computer Group in Madison, Wisconsin, and the failure parameters used (penalty for creation of space = 12, penalty for extension of space = 4) for that program can also be used to compare the similarity and identity of the sequence. "Antibodies" as used herein include polyclonal or monoclonal antibodies, chimeric, single-chain, and humanized antibodies, as well as antibody fragments (eg, Fab, Fab F (ab ') 2 and Fv), including Fab products or another 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 that do not substantially recognize and bind to other molecules in a sample containing a mixed population of antigenic biological molecules. Selection tests to determine the binding specificity of an antibody are well known and practiced routinely in the art. For a comprehensive discussion of such trials, see Harlow et al. (Eds.), ANTIBODIES TO LABORATORY MANUAL; Cold Spring Harbor Laboratory; Cold Spring Harbor, NY (1988), Chapter 6. The term "substantially pure" refers to a preparation comprising at least 50-60% by weight of the compound of interest (eg, nucleic acid, oligonucleotide, protein, etc.). ). More preferably, the preparation comprises at least 75% by weight, and more preferably 90-99% by weight, the compound of interest. Purity is measured by methods appropriate for the compound of interest (eg, chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like). With respect to single filament nucleic acid molecules, the term "specifically hybridizing" refers to the association between two single filament nucleic acid molecules of sufficiently complementary sequence to allow such hybridization under pre-determined conditions generally used in the matter (sometimes called "substantially complementary"). In particular, the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single strand RNA or DNA molecule, to the substantial exclusion of hybridization of the oligonucleotide with single strand 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 does not encode information related to protein synthesis. Such sequences are transcribed in 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 at the appropriate positions relative to the coding sequence to allow the expression of the sequence of coding. By way of example, a promoter is operably linked to a coding sequence when the promoter is capable of controlling the transcription or expression of that coding sequence. The 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 installation of other transcription control elements (e.g., enhancers) in an expression vector.

The transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, which provide for the expression of a coding sequence in a host cell. The terms "promoter", "promoter region" or "promoter sequence" generally refer to transcriptional regulatory regions of a gene, which may be found on the 5 'or 3' side of the coding region, or within the region of coding, or within introns. Typically, a promoter is a DNA regulatory region capable of binding the RNA polymerase in a cell and initiating the transcription of a coding sequence downstream (3 'direction). The typical 5 'promoter sequence is limited 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 detectable levels above previous. Within the pr.omotora sequence is a transcription initiation site (conveniently defined by tracing with nuclease SI), as well as protein binding domains (consensus sequences) responsible for RNA polymerase binding. A "vector" is a replicon, such as a plasmid, phage, cosmic, or virus to which another segment of nucleic acid can be operably inserted to cause 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 to transform a cell. This term may be used interchangeably with the term "transformation DNA" or "transgene". Such a nucleic acid construct can contain a coding sequence for a genetic product of interest, together with a selectable marker gene and / or reporter gene. A "marker gene" or "selectable marker gene" is a gene whose encoded genetic product confers a characteristic that allows a cell containing the gene to be selected from among cells that do not contain 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 resistance or tolerance genes, and (3) metabolic or autotrophic marker genes that allow transformed cells to synthesize an essential component, usually an amino acid, that the cells can not otherwise produce. A "reporter gene" is also a type of marker gene. Typically it encodes a genetic product that can be analyzed or detected by standard laboratory means (for example, enzymatic activity, fluorescence). The term "express", "expressed" or "expression" of a gene refers to the biosynthesis of a genetic product. The process includes the transcription of the gene into mRNA and then the translation of the mRNA into one or more polypeptides, and comprises all post-translational modifications that occur naturally. "Endogenous" refers to any constituent, for example, a gene or nucleic acid, or polypeptide, which may 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 largest molecule in nature. In this way, 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 by itself is not found in nature (eg, a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different from the native gene) . Allelic variations or mutational events that occur naturally do not give rise to a heterologous region of DNA as defined herein. The term "DNA construction", as defined above, is also used to refer to a heterologous region, particularly one constructed to be used in the transformation of a cell.

A cell has been "transformed" or "transfected" by heterologous or exogenous DNA when such DNA has been introduced into the cell. The transformation DNA may or may not be integrated (covalently linked) into the genome of the cell. In prokaryotes, yeast and mammalian cells for example, the transformation DNA can be maintained in an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transformation DNA has been integrated into a chromosome so that it is inherited by the daughter cells through the replication of the chromosome. 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 for many generations. "Grain", "seed", or "Jewish". refers to a plant unit in flowering reproduction, capable of developing in another such plant. As used herein, especially with respect to coffee plants, the terms are used synonymously and interchangeably. "Galactomannan synthesis enzyme" and "galactomannan synthesis gene" refers to a protein, or enzyme, and the gene encoding it, included in the synthesis of galactomannan polymers. Galactomannan synthesis enzymes include mannan synthases and galactosyl trans ferases. Similarly, galactomannan synthesis genes include genes encoding mannan synthases and galactosyl t rans ferases. As used herein, the term "plant" includes reference to whole plants, members of the plant (e.g., leaves, stems, shoots, roots), seeds, pollen, plant cells, plant cell organelles, and progeny of the same. The transgenic plant parts are to be understood within the scope of the invention comprising, for example, plant cells, prototypes, tissues, callus, embryos as well as flowers, stems, seeds, pollen, fruits, leaves, or roots that they originate in transgenic plants or their progeny. Description: Galactomanan is an abundant polysaccharide and a significant component of the mature coffee bean. Its great presence in the mature coffee bean supports the thought that its role is to maintain the integrity of the grain. Consistent with this, galactomannans, together with other saccharide components in coffee bean, are thought to play an important role in the extraction characteristics of coffee beans in water, which can affect the physical and chemical characteristics of the resulting coffee. The key enzymes included in the metabolism of this polysaccharide are galactomannan synthesis enzymes, such as mannan synthases and galactosyl trans fe rasas. One aspect of the present invention comprises coffee nucleic acid molecules that encode the synthases of mannan and galactose il trans ferases. cDNAs that encode a complete mannan synthase of Coffea ca n eph ora (pVC4, pVC6) are set forth herein as SEQ ID NOS: 1 and 2, respectively, and are referred to as CcMa n S. A cDNA encoding a full-length Coffea-a-bobbin synthase (pVC7) is set forth herein as SEQ ID NO: 3, and is referred to as CaMa n S. The partial genomic clones are set as SEQ ID NOS: 7, 8, 9 and 10, respectively, as discussed in the description of Figure 2A and in the examples. Additionally, the present nucleic acid molecules include cDNAs that encode galactose ilt rans ferases galatomannan, which in some cases are sequences that provide approximately 54% identity with a fenugreek galactosyltransferase, and in some cases sequences that provide about 54% identity with a galactosyltransferase from Japan icus. In some embodiments these cDNAs include the sequences provided in SEQ ID NOS: 11 or 13, which are referred to as CcGMGTl, and SEQ ID NOS: 12 or 14, referred to as CcGMGT2. Another aspect of the invention relates to proteins produced by the expression of these nucleic acid molecules and their uses The deduced amino acid sequences of the CcManS protein produced by the translation of SEQ ID NO: SEQ ID NO: 2 set forth herein as SEQ ID NOS: 4 and 5, respectively The deduced amino acid sequence of the CaManS protein produced by the translation of SEQ ID NO: 3 is set forth herein as SEQ ID NO: 6. The amino acid sequences deduced from the CcGMGTl protein produced by the translation of SEQ ID NO: 11 or SEQ ID NO: 13 are set forth herein as SEQ ID NOS: 15 and 17. The deduced amino acid sequences of the CcGMGT2 protein produced by the translation of SEQ ID NO: 12 or SEQ ID NO: 14 are set forth herein as SEQ ID NOS: 16 and 18. The table below lists the encoded proteins and polynucleotides referred to above, Polynucleotides and Pol. Iptides Included in the Galactomanan Synthesis Still other aspects of the invention relate to uses of the nucleic acid molecules and polypeptides encoded in the development of the plant and in genetic manipulation of the plants, and finally in the handling of properties of the coffee bean. Although the polynucleotides encoding the galactomannan synthesis enzymes of Coffea ca n ephra and Coffea a rabi ca are described and exemplified herein, this invention is intended to comprise nucleic acids and encoded proteins from other Co ffea species that are sufficiently similar to be used interchangeably with the proteins and polynucleotides of C. can ephora and Coffea for the purposes described below. Accordingly, when the galactomannan synthesis syntheses "mannan synthase" and galatomannan galactosyltransferase (or galactosyltransferase) are referred to herein, these terms are intended to comprise all mannan synthases of Coffea and galact osi 1 transferases having the general physical, biochemical and functional characteristics described herein, and polynucleotides encoding them, unless otherwise specifically stated .. Considered in terms of their sequences, the mannan synthase polynucleotides of the invention include variants allelic and natural mutants of SEQ ID NOS: 1-3, which are probably found in different varieties of C. ca n eph ora and Coffea a ra bi ca, and homologues of SEQ ID NOs: 1-3 are probably found in different. coffee species galactosyltransferase polynucleotides include allelic variants and natural mutants of SEQ ID NOS: 11-14, which They are found in different varieties of C. ca n eph ora and Coffea a rabi ca, and homologs of SEQ ID NOs: 11-14 are probably found in different coffee species. Because such variants and homologs are expected to possess certain amino acid and nucleotide sequence differences, there are nucleic acid molecules encoding the isolated mannan synthase and nucleic acid molecules encoding galactosyltransferase encoding respective polypeptides having at least about 75 % (and, with increasing order of preference, 76%, 77%, 78%, 79%, 70%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99%) of identity with the polypeptide encoded by SEQ ID NOS: 4, 5 or 6 in the case of mann synthases, and SEQ ID NOS: 15, 16, 17 or 18 in the case of galactosyltransferase. Because the variation of natural sequence probably exists between syn- theses of mannan and galact os i lt rans ferasas, and the genes that encode them in different varieties and species of coffee. 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 expectation is due in part to the degeneracy of the genetic code, as well as the known evolutionary success of conservative amino acid sequence variations, which do not appreciably alter the nature of the encoded protein. According to the foregoing, such variants and homologs are considered substantially equal to each other and are included within the scope of the present invention. The following sections establish the general procedures included to practice the present invention. to the extent that the specific materials are mentioned, it is merely for the purpose of illustration, and it is not proposed to limit the invention. Unless otherwise specified, general molecular and biochemical biological procedures are used, 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). . . .

Molecules of Nucleic Acid, Proteins and Antibodies: The nucleic acid molecules of the invention can be prepared by two general methods: (1) they can be synthesized from appropriate nucleotide triphosphates, or (2) they can be isolated from biological sources. Both methods use procedures well known in the art. The availability of information on the nucleotide sequence, such as the cDNA having SEQ ID NOS: 1-3 (or fragments represented by SEQ ID NOS: 7-10) or 11-14 (or fragments represented by SEQ ID NOS: 19 and 20) allows the preparation of an isolated nucleic acid molecule of the invention by oligonucleotide synthesis. Synthetic oligonucleotides can be prepared by the phosphoramidite method employed in the Applied Biosystems 38a DNA Synthesizer or similar devices. The resulting construct can 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 synthetic oligonucleotide methods. In this way, for example, a long double-stranded molecule can be synthesized as several smaller segments of appropriate complement. The complementary segments thus produced can be tempered so that each segment has appropriate cohesive terms for joining an adjacent segment. Adjacent segments can be ligated by quenching cohesive terms in the presence of DNA ligase to build a long, complete double-stranded molecule. A synthetic DNA molecule thus constructed can then be cloned and amplified into an appropriate vector. According to the present invention, nucleic acids having the appropriate level of sequence homology with part or all of the coding and / or regulatory regions of polynucleotides encoding galactomannan synthesis enzyme can be identified by using hybridization and rinsing conditions of appropriate severity. It will be appreciated by those skilled in the art that the aforementioned strategy, when applied to genomic sequences, in addition to allowing the isolation of enzyme coding sequences that metabolise the polysaccharide, will also allow the isolation of promoters and other genetic regulatory sequences associated with enzyme genes that metabolize polysaccharide, even when the regulatory sequences themselves may not share sufficient homology to allow adequate hybridization. As a typical illustration, hybridizations can be performed, according to the method of Sambrook et al. , using a hybridization solution comprising: 5X SSC, 5X Denhardt Reagent, 1.0% SDS, 100 μg / ml Denatured salmon sperm DNA, denatured, 0.05% sodium pyrophosphate and up to 50% formamide. Hybridization is carried out 37-42 ° C for at least six hours. After hybridization, the filters are rinsed as follows: (1) 5 minutes at room temperature in 2X SSC and 1% SDS; (2) 15 minutes at room temperature in 2X SSC and 0.1% SDS; (3) 30 minutes-1 hour at 37 ° C in 2X SSC and 0.1% SDS; (4) 2 hours at 45-55 ° C in 2X SSC and 0.1% SDS, changing the solution every 30 minutes. A common formula for calculating the severity 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 formula above, 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 each 1% decrease in homology. In this way, targets with greater than approximately 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 rinse is at 42 ° C; and in another embodiment the hybridization is at 42 ° C and the final rinse is at 50 ° C; and in yet another embodiment the hybridization is at 42 ° C and final rinsing is at 65 ° C, with the previous hybridization and rinsing solutions. High severity conditions include hybridization at 42 ° C in the above hybridization solution and a final rinse at 65 ° C in 0. IX SSC and 0.1% SDS for 10 minutes. The nucleic acids of the present invention can be maintained as DNA in any convenient cloning vector. In a preferred embodiment, the clones are maintained in plasmid cloning / expression vector, such as pGEM-T (Promega Biotech, Madison, Wl), pBluescript (Stratagene, La Jolla, CA), pCR4-T0P0 (Invitrogen, Carlsbad, CA) or pET28a + (Novagen, Madison, Wl), all of which can be propagated in a host cell E. col i adequate. The nucleic acid molecules of the invention include cDNA, genomic DNA, RNA, and fragments thereof which may be single, double or even triple filament. Thus, this invention provides oligonucleotides (sense or antisense filaments 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 the galactomannan synthesis enzyme or mRNA in plant tissue test samples, for example, by PCR amplification, or for the positive or negative regulation of expression of genes encoding the enzyme of galactomannan synthesis in or before translation of mRNA in proteins. The methods in which the polynucleotides or oligonucleotides encoding the galactomannan synthesis enzyme can be used as probes for such analyzes include, but are not limited to: (1) hybridization i n s i t u; (2) Southern hybridization; (3) Northern hybridization; and (4) affirmed classification reactions such as polymerase chain reactions (PCR, including RT-PCR) and ligase chain reaction (LCR). Oligonucleotides having sequences capable of hybridizing to at least one sequence of a nucleic acid molecule of the present invention include antisense oligonucleotides. Antisense oligonucleotides that are targeted to specific regions of the mRNA that are critical can be used. The use of antisense molecules to lower the levels of expression of a pre-terminated gene is known in the art. Antisense molecules can be provided i n s i t u by transforming plant cells with a DNA construct that, in transcription, produces the antisense RNA sequences. Tale.s.construccióne.s can be designed to produce full-length or partial antisense sequences. This gene silencing effect can be enhanced by transgenerationally overproducing both sense and antisense RNA from the genetic coding sequence so that a high amount of dsRNA is produced (e.g., see Waterhouse et al., 1998, PNAS 9_5: 13959-13964 ). In this regard, dsRNA containing sequences that correspond to part or all of at least one intron has been found to be particularly effective. In one embodiment, part or all of the sequence antisense filament encoding galactosyltransferase or mannan synthase is expressed by a transgene. In another embodiment, the sense and antisense filaments of hybridization of part or all of the sequence encoding galactosyltransferase or sequence encoding mannan synthase are transgene expressed. In another embodiment, the genes of mannan synthase or galactosyltransferase genes or both can be silenced by the use of * small interfering RNA (siRNA; Elbashir et al., 2001, Genes Dev. 15 (2): 188-200) using materials and methods commercially available. (for example, Invitrogen, Inc., Carlsbad CA). The polypeptides encoded by the nucleic acids of the invention can be prepared in a variety of ways, according to known methods. If they occur, the polypeptides can be purified from appropriate sources, for example, seeds, pericarps, or other parts of the plant. Alternatively, the availability of nucleic acid molecules encoding the polypeptides allows the production of the proteins using expression methods known in the art. For example, a cDNA or gene can be cloned into an appropriate transcription vector in vi t ro, such as pSP64 or pSP65 for transcription in vi t ro, followed by cell-free translation in a suitable cell-free translation system, such as Wheat germ or rabbit reticulum. Translation and transcription systems are commercially available, for example, from Promega Biotech, Madison, WI, BRL, Rockville, MD or Invitrogen, Carlsbad, CA. According to a preferred embodiment, larger amounts of polypeptides can be produced by expression in a suitable eukaryotic or prokaryotic system. For example, part or all of a DNA molecule, such as the cDNA having SEQ ID NO: 2 or SEQ ID NO: 3 or any of SEQ ID NOS: ll-14, can be inserted into a plasmid vector adapted for expression in a bacterial cell (such as E. co li) or a yeast cell (such as Sa ech a romyee s cerevi siae), or in a baculovirus vector for expression in an insect cell. Such vectors comprise the regulatory elements necessary for the expression of DNA in the host cell, placed in such a manner to allow expression of the DNA in the host cell. Such regulatory elements required for expression include promoter sequences, transcription initiation sequences and, optionally, enhancer sequences. Polypeptides produced by gene expression in a recombinant eukaryotic or prokaryotic system can 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 then secreted from the host cell, and then purified from the surrounding medium. An alternative approach includes purifying the recombinant protein by affinity separation, for example, through immunological interaction with antibodies that specifically bind to the recombinant protein. The polypeptides of the invention, prepared by the above-mentioned methods, can be analyzed according to standard procedures. The purified polypeptides of coffee or recombinantly produced can be used to generate polyclonal or monoclonal antibodies, antibody fragments or derivatives as defined herein, according to known methods. In addition to making antibodies to the complete recombinant protein, if analyzes of proteins or Southern and cloning analyzes (see below) indicate that the cloned genes belong to a multigen family, then the member-specific antibodies made for synthetic peptides corresponding to regions not preserved proteins can be generated. . _ _. . -. The 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 equipment includes a control antigen for which the antibody is immunospecific. Vectors, Cells, Tissues and Plants: Vectors and kits for producing transgenic host cells containing an oligonucleotide or polynucleotide encoding the galactomannan synthesis enzyme, or variants thereof in a sense orientation are also characterized in accordance with the present invention. or antisense, or reporter gene and other constructs under the control of enzyme promoters that metabolize the polysaccharide 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 skilled in the art. They include, but are not limited to, plasmids, cosmids ,. baculoviruses, bacmides, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), as well as other bacterial, yeast and viral vectors. Typically, the kits for producing transgenic host cells will contain one or more appropriate vectors and instructions for producing the transgenic cells using the vector. The kits may also include one or more additional components, such as culture medium to grow the cells, reagents for transforming the cells and reagents to test the transgenic cells for gene expression, to name a few. The present invention includes transgenic plants comprising one or more copies of the gene encoding the galactomannan synthesis enzyme, or nucleic acid sequences that inhibit the production or function of an endogenous galactomannan synthesis enzyme from the plant. This is done by transforming the plant cells with a transgene comprising part of a galactomannan synthesis enzyme, or mutant, antisense or variant thereof, including RNA, controlled by either native or recombinant regulatory sequences. , as described below. Coffee species of transgenic plants are preferred, including without limitation, C. abeokutae, C. arabica, C. arnoldiana, C. aruwemiens is, C. bengalensis, C. canephora, C. congens is, C. Dewevrei, C. excelsa, C. eugenioides, and C. heterocalyx, C. kapakata, C. khas iana, C. 1 Iberica, C. moloundou, C. ra semosa, C. salvatrix, C. sessi flora, C. stenophylla, C. t avencorens is, C. wightiana and C. zanguebar iae. Plants of any species are also included in the invention; These include, but are not limited to, tobacco, Arabidops and other "laboratory friendly" species, cereal crops such as corn, wheat, rice, soybean barley, rye, oats, sorghum, alfalfa, clover and similar, plants that produce oil such as cañola, safflower, sunflower, peanut, cocoa and the like, vegetable crops such as tomatillo, potato, pepper, eggplant, sugar beet, carrot, cucumber, lettuce, pea and the like; such as asters, begonia, chrysanthemum, delphinium, petunia, zinnia, grass and herbs and the like Transgenic plants can be generated using standard plant transformation methods known to those skilled in the art, including, but not limited to. a, Agroba ct eri um vectors, Protoplast polyethylene glycol treatment, biolistic DNA supply, UV laser microbeam, germ virus vectors or other viral vectors of plant, treats Calcium phosphate treatment of protoplasts, electroporation of isolated protoplasts, agitation of cell suspensions in solution with microbeads coated with the transformation DNA, agitation of cell suspension in solution with silicon fibers coated with DNA transformation, DNA uptake direct, DNA capture mediated by liposome, and the like. Such methods have been published in the subject. See, for example, Methods for Plant Molecular Biology (Weissbach &Weissbach, eds., 1988); Methods in Plant Molecular Biology (Schuler &Zielinski, eds., 1989); Plant Molecular Biology Manual (Gelvin, Schi lperoort, Verma, eds., 1993); and Methods Xn Plant Molecular Biology-A Laboratory Manual (Maliga, Klessig, Cashmore, Gruissem &Varner, eds., 1994). The transformation method depends on the plant to be transformed. Agroba c t eri um vectors are often used to transform dicot species. The Agroba ct eri um binary vectors include, but are not limited to, BIN19 and derivatives thereof, the pBI vector series, and the binary vectors pGA482, pGA492, pLH7000 (Access GenBank AY234330) and any suitable vectors pCAMBIA ( derived from the pPZP vectors constructed by Ha jdukiewicz, Svab &Maliga, (1994) Plant Mol Biol 25: 989-994, available from CAMBIA, GPO Box 3200, Canberra ACT 2601, Australia or via the worldwide network at CAMBIA.org) . For the transformation of monocot species, biolistic bombardment with particles coated with transformation DNA and silicon fibers coated with transformation DNA are often useful for nuclear transformation. Alternatively, the "superbinar ios" Agroba ct eri um vectors have been used successfully for the transformation of rice, maize, and several other monocot species. "DNA constructs for transforming a selected plant comprise a coding sequence of interest operably linked to appropriate 5 'sequencing (e.g., promoters and translation regulatory sequences) and 3' regulatory sequences (e.g., terminators). , the sequences encoding the galactomannan synthesis enzyme under the control of their own 5 'and 3' own elements can be used In other embodiments, the regulatory and coding sequences of the galactomannan synthesis enzyme are changed to alter the profile of the polysaccharide of the transformed plant for a phenotypic improvement, for example, in taste, aroma or other characteristic, such as coffee foam produced In an alternative embodiment, the coding region of the gene is placed under a powerful constitutive promoter, such as 35S promoter from Cauliflower Mosaic Virus (CaMV) or the 35S promoter of the esopharyngeal mosaic virus ia. Other constitutive promoters contemplated for use in the present invention include, but are not limited to, T-DNA mannopine synthase, nopaline synthase, and octopine synthase promoters. In other embodiments, a strong monocot promoter is used, for example, the corn ubiquitin promoter, the rice actin promoter, or the rice tubulin promoter (Jeon et al., Plant Physiology, 123: 1005-14). , 2000).

Transgenic plants expressing galactomannan synthesis enzyme coding sequences under an inducible promoter are also contemplated to be within the scope of the present invention. Inducible plant promoters include tetracycline repressor / operator-controlled promoter, heat shock genetic promoters, stress-induced promoters (eg, wound), defense-responsive genetic promoters (eg, ammonium lyase genes of phenylalanine), wound-induced genetic promoters (e.g., hydroxyproline-rich cell wall protein genes), chemically-inducible gene promoters (e.g., nitrate reductase genes, glucanase genes, quintinase genes, etc.) and dark-inducible genetic promoters (eg, asparagine synthetase gene) to name a few. Developmental-specific and tissue-specific promoters are also contemplated for use in the present invention. Non-limiting examples of seed-specific promoters include Ciml (message induced by cytoquinoline), cZ19Bl (corn 19kDa zein), milpas (myo-inosi synthase tol-1-phosphate), and celA (cellulose synthase) ( U.S. Application Ser. No. 09 / 377,648), bet a- bean phaseollin, napkin, beta-conglycinin, soybean lectin, cruciferite, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein , waxy, shrunken 1, shrunken 2, and globulin 1, soybean legume 11S (Báumlein et al., 1992) and seed storage protein of C. ca n eph ora US (Marraccini et al., 1999). See also WO 00/12733, where the preferred seed promoters of the end 1 and end 2 genes are described. Other Coffea seed specific promoters can also be used, including but not limited to the oleosin gene promoter described in the commonly co-pending PCT Application in property No. US2006 / 026121, the dehydrin gene promoter described in the PCT Application co. commonly assigned in property No. US2006 / 026234, and the 9-cis-epoxycarotenoid dioxygenase gene promoter described in the co-pending PCT Application, commonly owned No. US2006 / 034402. Examples of other tissue-specific promoters include, but are not limited to: promoters of small subunit gene of ribulose bisphosphate carboxylase (RuBisCo) (eg, the small coffee subunit promoter as described by Marracini et al., 2003) or promoters of the protein gene of chlorophyll a / b (CAB) binding for the expression of photosynthetic tissue; and the specific root-specific glutamine synthetase promoters where root expression is desired. The coding region is operably linked to an appropriate 3 'regulatory sequence. In embodiments where the native 3 'regulatory sequence is not used, the region of polyadenylation of nopaline synthetase can be used. Other useful 3 'regulatory regions include, but are not limited to, the octopine synthase polyadenylation region. The selected coding region, under the 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 resistance to herbicide or antibiotic (e.g., hygromycin resistance, sulfonylurea, fos ricot, or glyphosate) or genes that confer selective growth (eg, phosphomannose isomerase, allowing the growth of plant cells in mannose). Selectable marker genes include, without limitation, genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO), dihydrofolate reductase or (DHFR) and hygromycin phosphotransferase (HPT), as well as genes which confer resistance to herbicidal compounds, such as glyphosate-resistant EPSPS and / or glyphosate oxidoreductase (GOX), Brom oxyn i 1 nit ri lase (BXN) for bromoxynil resistance, AHAS genes for imidazole resistance, inonas, resistance genes sulfonylurea, and genes of resistance to 2,4-dichlorophenoxycetate (2,4-D). In certain embodiments, the promoters and other expression regulatory sequences encompassed by the present invention are operatively linked to reporter genes. Reporter genes contemplated for use in. The invention includes, but is not limited to, genes encoding green fluorescent protein (GFP), red fluorescent protein (DsRoja), Fluorescent Cyan Protein (CFP), Yellow Fluorescent Protein (YFP), Orange Fluorescent Protein Cerianthus (cOFP), alkaline phosphatase (AP), β-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), dihydrofolate reductase (DHFR) of aminoglycoside phosphotransferase (neo1, G418r), hygromycin-B-phosphotransferase (HPH) ), thymidine kinase (TK), lacZ (coding a-galact osidase), and fos for ibos i 1 guanine xanthine trans ferase (XGPRT), Bet a-Glucuronidase (gus), Placental Alkaline Phosphatase (PLAP), Secreted Embryonic Alkaline Phosphatase (SEAP), or Bacterial Luciferase or firefly (LUC). As with many of the standard procedures associated with the practice of the invention, experts will be aware of the additional sequences that can serve the function of a marker or reporter. Additional sequence modifications are known in the art to improve gene expression in a cellular host. These modifications include the elimination of sequences encoding superfluous polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well-characterized sequences that may be harmful to gene expression. Alternatively, if necessary, the G / C content of the coding sequence can be adjusted to the average of levels for a coffee plant cell host, as calculated for reference to known genes expressed in a coffee plant cell. Also when possible, the coding sequence is modified to avoid the predicted secondary pin mRNA structures. Another alternative to improve gene expression is to use 5 'leader sequences. Translation leader sequences are well known in the art, and include the cis (omega ') derivative of the 5' (omega) guide sequence of tobacco mosaic virus, the 5 'guide sequences of bromine mosaic virus , Alphafa mosaic Xirus, and yellow turnip mosaic virus. The plants are transformed and then selected for one or more properties, including the presence of the transgenic product, mRNA encoding transgene, or an altered phenotype associated with the expression of the transgene. It should be recognized that the amount of expression, as well as the specific temporal and tissue pattern of transgene expression in transformed plants may vary depending on the position of their insertion in the nuclear genome. Such position effects are well known in the art. For this reason, several nuclear transformers must be regenerated and tested for transgene expression. Methods: The nucleic acids and polypeptides of the present invention can be used in any of a number of methods whereby the production of protein products in coffee plants can be modulated to affect various phenotypic treatments, for example, for improving the flavor, foam (physical property) and / or aroma of the beverage of coffee or coffee products produced last of the grain, or for improvement in the qualities of production of the grains. For example, a decrease in galactomannan content, or an alteration of g. Lactomannan structure, is expected to greatly improve the recovery of solids in the galactomannan structure process. The improvement of the coffee grain polysaccharide profile or other characteristics can be obtained by (1) classical breeding or (2) genetic engineering techniques, and by combining these two approaches. Both approaches have been considerably improved by the isolation and characterization of a gene encoding the galactomannan synthesis enzyme in coffee, according to the present invention. For example, the genes encoding the galactosyltransferase or synthase can be traced genetically and Quantitative Trait Places (QTL) included in coffee flavor can be identified. It would then be possible to determine whether such QTL correlates with the position of genes related to galactosyltransferase or mannan synthase. The alleles (haplotypes), for genes that affect the metabolism of the polysaccharide can also be identified and examined to determine if the presence of specific haplotypes correlates strongly with galactomannan synthesis. These markers can be used to take advantage in assisted parenting programs. A third advantage of isolating the polynucleotides included in galactomannan synthesis is to generate the expression data for these genes during maturation of the coffee bean in varieties with high and low levels of galactomannan. This information can be used to direct the choice of genes for use in assisted genetic manipulation to generate new transgenic coffee plants that have increased or decreased galactomannan levels in the mature grain. In one aspect, the present invention comprises methods for altering the galactomannan profile in a plant, preferably coffee, comprising increasing or decreasing an amount or activity of one or more galactomannan synthesis enzymes in the plant. The specific embodiments of the present invention provide methods to increase or decrease the production of morning synthase. In one embodiment coffee plants can be transformed with a polynucleotide encoding mannan synthase, such as a cDNA comprising SEQ ID NO: 2 or 3, or 11-14, for the purpose of over-producing mannan synthase or galactosyltransferase , respectively, in various coffee tissues. In one embodiment, the coffee plants are manipulated for a general increase in mannan synthase production, for example, through the use of a promoter such as the RuBisCo small subunit promoter (SSU) or the CaMV35S promoter functionally linked to a Manan synthase gene. In another embodiment, coffee plants are engineered for a general increase in galactosyltransferase production, for example, through the use of a promoter such as the RuBisCo small subunit promoter (SSU) or the CaMV35S promoter functionally linked to a galactosyltransferase. In some embodiments, the modification of coffee plants can be manipulated to increase both the mannan synthase and the galactosyltransferase production. In another embodiment designed to limit the production of mannan synthase, or galactosyltransferase, only to the diminished member of interest, ie, the grain, can a specific grain promoter be used, particularly one of the specific promoters of Co-ffea grain. described above Plants that display altered galactomannan profiles can be selected for naturally occurring variants of mannan synthase or galactosyltransferase For example, loss-of-function (null) mutant plants can be created or selected from populations of plant mutants Currently available It will also be appreciated by those skilled in the art that mutant plant populations can also be selected for mutants that sub-overexpress a particular polysaccharide-enhancing enzyme., such as a galactomannan synthesis enzyme, using 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 target local lesions induced in genomes (TILLING, see, eg, Henikoff et al., 2004, Plant Physiol. (2): 630-636; Gilchrist & Haughn, 2005, Curr. Opin. Plant. Biol. 8 (2): 211-215). Methods for making mutant populations are well known in the art. The nucleic acids of the invention can be used to identify mutant forms of galactomannan synthesis enzyme in various plant species. In species such as corn or Arabi dops i s, where the transposon insertion lines are available, the oligonucleotide primers can be designed to select lines for insertions in the galactomannan synthesis enzymes. Through breeding, a plant line can then develop which is heterozygous or homozygous for the interrupted gene. A plant can also be manipulated to display a phenotype similar to that observed in null mutants created by mutagenic techniques. A transgenic null mutant can be created by expressing a mutant form of galactomannan synthesis enzyme to create a "dominant negative effect". Although not limiting the invention to any mechanism, this mutant protein will compete with wild-type protein to interact with proteins or other cellular factors. Examples of this type of "dominant negative" effect are well known for both vertebrate and insect systems (Radke et al., 1997, Genetics 145: 163-171; Kolch et al. , 1991, Nature 349: 426-428). .. . Another class of transgenic null mutant can be created by inhibiting the translation of mRNA that encodes the galactomannan synthesis enzyme by "silencing the post-t-translation gene". These techniques can be used to sub-regulate the mannan synthase in a plant bean, thus altering the profile of the polysaccharide. For example, a gene encoding the galactomannan synthesis enzyme of the target species for sub-regulation, or a fragment thereof, can be used to control the production of the encoded protein. The full-length antisense molecules can be used for this purpose. Alternatively, the antisense oligonucleotides targeted to specific regions of the mRNA that are critical for translation may be used. The use of antisense molecules to lower the expression levels of a predetermined gene is known in the art. Antisense molecules can be provided i n s i t u by transforming plant cells with a DNA construct that, in transcription, produces the antisense RNA sequences. Such constructions can be designed to produce. partial or full-length antisense sequences. This gene silencing effect can be enhanced by transgenerationally overproducing both sense and antisense RNA of the gene coding sequence so that a high amount of dsRNA is produced (e.g., 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 has been found to be particularly effective. In one embodiment, part or all of the sequence antisense strand encoding mannan synthase is expressed by a transgene. In another embodiment, part or all of the sequence antisense filament encoding mannan synthase is expressed by a transgene. In another embodiment, genes encoding galactomannan synthesis can be silenced through the use of a variety of other posttranslational gene silencing (RNA silencing) techniques that are currently available for plant systems. RNA silencing involves the processing of double-stranded RNA (dsRNA) in 21-28 small nucleotide fragments by an enzyme based on RNase H ("Dicer" or "similar to Dicer"). 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 RNA silencing reviews in plants, see Horiguchi, 2004, Difference 7_2: 65-73, Baulcombe, 2004, Nature 431: 356-363, Herr, 2004, Biochem Soc. Trans. 3_2_: 946-951). Small interfering RNAs can be synthesized or chemically transcribed and amplified in vitro, and then delivered to the cells. The supply can be through microinjection (Tuschl T et al., 2002), chemical transfection (Agrawal N et al., 2003), elect oporation or transfection mediated by cationic liposome (Brummelkamp TR et al., 2002; Elbashir SM et al., 2002), or any other means available in the matter, which will be appreciated by the expert. Alternatively, siRNA can be expressed intracellularly by inserting DNA siRNAs into the cells of interest, for example, by means of a plasmid, (Tuschl T et al., 2002) and can specifically target cells. Small interference RNAs have been successfully introduced into plants (Klahre U et al., 2002).

A preferred method of silencing RNA in the present invention is the use of short pin RNAs (shRNA). A vector containing a DNA sequence encoding a particular desired siRNA sequence is delivered to a target cell by a common means. Once in the cell, the DNA sequence is continuously transcribed into RNA molecules that surround themselves and form pin structures through formation in intramolecular base pairs. These pin structures, once processed by the cell, they are equivalent to siRNA molecules and are used by the cell to mediate RNA silencing of the desired protein. Several constructs of particular utility for RNA silencing in plants are described by Horiguchi, 2004, s upra. Typically, such a construct comprises a promoter, a target gene sequence to be silenced in the "sense" orientation, a spacer, the antisense of the target genetic sequence, and a terminator. Still another type of synthetic null mutant can also be created by the "co-suppression" technique (Vaucheret et al., 1998, Plant J. 16 (6, 651-659) Plant cells are transformed with a copy of the target endogenous gene for repression In many cases, this results in complete repression of the native gene as well as the transgene In one embodiment, a gene encoding enzyme of galactomannan synthesis of the plant species of interest is isolated and used to transform cells of those same species Transgenic or mutant plants produced by any of the above methods are also characterized according to the present invention .Preferably, the plants are fertile , thus being useful for breeding purposes, in this way, mutants or plants that show one or more of the desirable phenotypes mentioned above can be used for plant breeding, or directly in agricultural or horticultural applications. as search tools for the additional production of enzymes participation I t abol i zant e s of polysaccharide and its affections in polysaccharide profiles, thus affecting the taste, aroma and other characteristics of coffee seeds.

Plants that contain a specific transgene or mutation can also be crossed with plants that contain a complementary genotype or transgene to produce plants with combined or improved phenotypes. The following examples are provided to describe the invention in greater detail. The examples are for illustrative purposes, and are not intended to limit the invention. Example 1 Materials and Methods for Subsequent Examples of Plant Material. Cherry trees (BP409, 2001) are harvested from trees in the field at the Indonesian Coffe and Cacao Research Center (ICCRI), Indonesia. Immediately after harvesting, the cherry trees are frozen in liquid nitrogen and then shipped frozen in dry ice to the designated location for further processing. Samples are frozen at -25 ° C for transportation, then stored at -80 ° C until use. DNA sequence analysis. For DNA sequencing, recombinant plasmid DNA is prepared and sequenced according to standard methods. The computer analysis is done using DNA Star software (Lasergene). Sequence homologies are verified against GenBank databases using BLAST programs (Altschul et al., 1990). Preparation of cDNA. cDNA is prepared from Total RNA and oligo dT (18) (Sigma) as follows: 1 μg sample of total RNA plus 50 ng oligo dT is made up to 12 μl final volume with water treated with DEPC. This mixture is subsequently incubated at 70 ° C for 10 min and then cooled rapidly on ice. Then, 4 μl of the first filament regulator (5x, Invitrogen), 2 μl of DTT (0.1M, Invitrogen) and 1 μl of dNTP mixture (10 mM each, Invitrogen) are added. These reaction mixtures are pre-incubated at 42 ° C for 2 min before adding 1 μl H-Reverse Transcriptase SuperScript III Rnase (200U / μl, Invitrogen). Subsequently, the tubes are incubated at 25 ° C for 10 min and then at 42 ° C for 50 min, followed by inactivation of enzyme upon heating at 70 ° C for 10 min. The generated cDNA samples are then diluted ten times in sterile water and stored at -20 ° C for use in some of the following experiments, such as RACE 5 ', isolating full-length cDNA clones, and QRT-PCR. 5 'RACE Reactions (Rapid Amplification of cDNA Ends) To recover the 5' coding sequence of the coffee manan synthase, two turns of RACE 5 'are carried out. The RNA used for the synthesis of cDNA in 5 'RAZA experiments is Coffea canephora grain (BP409) in the yellow stage. The RAZA 5 'experiments are carried out using methods that closely follow the methods described in the equipment for the RAZA 5' system for Rapid Amplification of cDNA End Equipment (Invitrogen). Briefly, the cDNA used in this experiment is first purified to remove any unincorporated nucleotides (since they would interfere with the subsequent DC reaction). This step is carried out by purifying the RAZA 5 'cDNA in columns S.N.A.P (Invitrogen) precisely according to the instructions given by the manufacturer. Once purified, the cDNA is recovered in 50 μL of sterilized water and then stored at -20 ° C before being used for PCR RAZA 5 '. The 5 'RAZA experiments all start with a subsequent TdT of the cDNA purified by SNAP.The subsequent poly dC reaction was as follows: reactions of 25 μl are established with 5 μl of the purified cDNA, 11.5 μl DEPC treated with water, μl 5x regulator posterior TdT (Invitrogen), and 2.5 μl 2 mM dCTP. The reactions are then incubated at 94 ° C for 3 minutes, followed by cooling on ice. 1 μl of TdT is then added and the reaction is incubated for 10 minutes at 37 ° C. The reactions are terminated by heating 10 minutes at 65 ° C and again placed on ice. The first round of RAZA 5 'reactions is performed in a final volume of 50 μl, as follows: 5 μl of each subsequent cDNA, 5 μl 10 x PCR regulator (ThermoPol regulator), 400 nM of both Gen 1 Specific Primer and primers AAP (see Tables 1 and 2 for primers), 200 μM each dNTP, and 2.5 U Taq DNA polymerase (BioLabs). First cycle PCR cyclization conditions were: 94 ° C for 2 min; then 40 cycles of 94 ° C for 1 min, tempering temperature observed in Table 2 for 1 min, and 72 ° C for 2 min for 40 cycles. An additional final stage of elongation is made at 72 ° C for 7 min. The PCR products are then analyzed by agarose gel electrophoresis and ethidium bromide staining. The second round PCR reactions are performed in a final volume of 50 μl, as follows: 5 μL of 1% diluted first round PCR product; 5 μl 10 x PCR regulator (LA II Mg ++ plus regulator), 200 nM of both Gen 2 Specific Primer and AUAP primers (see Tables 1 and 2 for specific primers used), 200 μM each dNTP, 0.5 U of Takara DNA polymerase LA Taq (Cambrex Bio Science). The cycle procedure was: 94 ° C for 2 min; then 40 cycles of 94 ° C for 1 min, the tempering temperature observed in Table 2 for 1 min, and 72 ° C for 1 min 30 seconds. An additional final stage of elongation is made at 72 ° C for 7 min. The PCR products are then analyzed by agarose gel electrophoresis and ethidium bromide staining.

Table 1. List of primers used for RAZA 5 'PCR experiments Table 2 Primers and PCR conditions used for the different RAZA 5' experiments The primers, tempering temperatures, and the number of cycles are given for the various PCR reactions RAZA 5 The sequences of DNA primers are given above, Table 1 Isolation of cDNA containing the complete coding sequences (complete ORF's) for ManS from coffea canephora and arabica coffea using gene-specific primers. The existing cDNA sequences, and the new 5 'sequences obtained from RAZA 5', are used to design 2 gene-specific primers in the 5 'and 3' UTR sequences to amplify the entire ORS sequences of ManS (pVC4, pVC6 and pVC7). The cDNA used to isolate the entire ORF sequences are shown in Table 3 (Seed, yellow stage, BP409, and Seed, yellow stage, T2308), and the sequences of the specific primers for each PCR reaction are given in Table 4 PCR reactions are performed in 50 μl reactions as follows: 5 μL of cDNA (Table 3 and 4), 5 μl 10 x PCR regulator (Regulator PCR II Mg + + plus), 800 nM of each gene-specific primer , 200μM of each dNTP, and 0.5 U of DNA polymerase Takara LA Taq (Cambrex Bio Science). After denaturing at 94 ° C for 2 min, the amplification consisted of 35 cycles of 1 min at 94 ° C, 1 min 30 seconds at annealing temperature (47 ° C) and 3 min at 72 ° C. An additional final stage of elongation is made at 72 ° C for 7 min. The PCR products are then analyzed by agarose gel electrophoresis and ethidium bromide staining. Fragments of the expected size are then cloned into pCR4-TOPO using TOPO TA Cloning Kit for Sequencing (Invitrogen) according to the instructions given by the manufacturer. The insertions of the generated plasmids are then completely sequenced. Gene CADN tissue and specific primer Tempe ra ur a gen gen tem tem gen CcMa n S Bp409 ManS-Am3 / mSS-m2 47 ° C Semiyla, lid loves r 111 a CaMa n S C. a ra bi ca T2308 ManS-Am3 / M nS-Am2 47 ° C Semília, amaman stage Table 3. Isolation of cDNA sequences encoding the full-length protein sequences for Manan's Sintasa from Coffea canephora (CcManS) and Manan's Synthase from Coffea arabica (CaManS) The specific cDNA, primers, and PCR tempering temperatures used to amplify the sequences of complete ORF are presented. These cDNAs are synthesized as described in the methods.

Table 4. Sequences of the primers used for the amplification of cDNA sequences encoding the full-length protein sequences of CcManS and CaManS Analysis of CcManS expression using quantitative RT-PCR (Q-RT-PCR) The cDNA used for these experiments is prepared according to the methods described above (robust, C. ca n eph ora BP 409 1/1000 dilution, sample cDNA arabica, C. a rabi ca T-2308 1/1000 dilution, cDNA sample). TaqMan-PCR is performed as recommended by the manufacturer (Applied Biosystems, Per kin-Elmer). Briefly, 25-ul reactions are established in the reaction plates (96-well Reaction Plate, MicroAmp Applied Biosystems, ref: N-801-0560). Each reaction contained 12.5 ul of AmpliTaq Gold Master mix, 2.5 ul of the two primers (8uM of rserva, 800nM final in reaction), 2.5 ul of MGB TaqMan probe (2uM of reserve, final 200nM in reaction), and 5 ul of sample of DNA plus water. The water and DNA is added to the plates first, then the "Specific Mix" (AmpliTaq Gold Master mix + primers and TaqMan probe) is added. The reactions are done at room temperature and the Taq amplifications begin only when Taq is activated by releasing the antibody bound at high temperatures, ie, Hot Start. The Taiman regulator contains AmpErase® UNG (Uracil-N-glycosylase), which is active for the first 2 min at 50 ° C and then activated at 95 ° C at the start of the PCR cycle. The cycle conditions used (Real Time PCR System 7500-Applied Biosystems) were 50 ° C 2 minutes, 95 ° C 10 minutes, then 40 cycles of 95 ° C 15 seconds and 60 ° C 1 minute. Each reaction is done in triplicate and the average Ct value for the three reactions is calculated. The used TaqMan primers and probes are designed with the PRIMER EXPRESS software (Applied Biosystems). The primers and MAB synthase probe used for Q-PCR experiments are 124613-F1 124613-R1 and 124613MGB1 (see Table 5). The quantification is carried out using the relative quantification method (RQ), using the gene of "ribosomal protein of coffee constitutively expressed CcRpl39 as the reference." In this case, average Ct is calculated for the genes CcManSyn (gene test) and .CcRpl39 (reference gene) of the replicates made for each gene in each tissue sample The RQ value (2-deitact with delta ct = CcManS Ct-CcRpl39 Ct), which is a measure of the difference between the two samples , then calculated To use the relative quantization method, it is necessary to show that the amplification efficiency for the test gene is equivalent to the amplification efficiency of the reference sequence (rp l 39 cDNA sequence) using the primer sets and Specified probe (amplification efficiency close to 1, ie 100%) To determine this relative equivalence, plasmid DNA containing the appropriate cDNA sequences are diluted 1/1000, 1 / 10,000, 1 / 100,000 and 1 / 1,000,000 times, and using the Q-PCR conditions described above, the slope of the curve Ct = f (DNA Log Amount) is calculated for each set of plasmid / primer / TaqMan probe. The TaqMan plasmid / primer / probe sets giving curves with slopes close to 3.32, representing an efficiency of "100%, are considered acceptable.The Taqman plasmid / primer / probe assemblies used here have acceptable values for Ct = f (Amount DNA log).

Table 5. List of primers used for Q-PCR experiments Example 2 Identification of Manan Synthase Coding for cDNA in C. canephora More than 47,000 EST sequences are identified from several coffee libraries made with RNA isolated from young leaves and from the pericarp grain and tissue of cherry trees harvested at different stages of development. Coating ESTs are subsequently "grouped" into "unigenes" (i.e., contiguous) and the sequences of unigen are annotated by performing a BLAST search of each individual sequence against the non-redundant protein database NCBI. Galactomannans contribute greatly to the dry weight of the mature coffee bean and are thought to play an important role in accessing or extracting molecules within the bean, for example, sugars. Methods are taken to isolate one of the key genes included in galactomannan synthesis, ie, mannan synthase, and study the expression of this gene to develop coffee bean. This sequence of mann synthase proteins biochemically characterized from guar (CtManS, Cya n ops istet ra gon ol oba, access number AAR23313; Dhugga et al., 2004) is used to search our 'unigen' set of cDNA sequences using the tblastn algorithm (Altschul, et al., 1990). This search discovered a unigen with a very high level of homology (unite # 124613). See Table 6. The two longest ESTs in this unite are completely isolated and sequenced: one, the fish insertion 46wl 6i 11, is found to be 1779 bp long; while the second, an insertion in fish 6w2 cl 9, is found to be 1349 bp long. An alignment analysis between these two sequences indicated that two intron sequences exist in the 46wl 6i 11 fish cDNA. As graphically observed in Figure 2A, one of the introns was the 5 'end of this clone, while the other, much smaller, the intron sequence is buried in the ORF of the cDNA. When the intron sequences are spliced out of the consensus sequence for these two cDNA clones, a partial ORF of 423 amino acids was discovered; however, the full length guar protein is 526 amino acids long. In this way, coffee ManS cDNA is not complete and lacks over 309 base pairs (ie, encoding 103 amino acids plus 5 'UTR).

Table 6. In S i l i co of Coffee Well Synthesis ESTs.

The number of manned synthase ESTs (ungen 124613) found in each of the different EST libraries of Co ffe a ca n eph EXAMPLE 3 Full-length ManS Sequence The fish clone 46wl 6i 11 encodes a significant portion of the coffee ManS sequence, thus, it is used to designate specific primers for use in the well-established genome-assisted genome walking technique. The first experiment produced a fragment 1084 bp long (pJMc2), which lengthens the introhica region by an additional 1000 bp more. However, since the new sequence does not contain any sequence information in the next .exon, this fragment does not produce any new sequence data in the ORF. The additional genome walking experiments do not generate new upstream sequences. PCR RAZA 5 ', as described in Example 1, is carried out to isolate the missing 5' coding region of this gene. This is done using gene-specific primers RNAi-Pr2-GSP1 and ManSynt GWR249-GSP2. The result was a PCR fragment of 300 base pairs, which is cloned into the vector pCR-4-TOPO and then sequenced. The obtained sequence (pVC2; CcManS Razal) was 259 bp long and covers the 5 'end of the fish cDNA clone 46wl 6 i 11 (Figure 2, showing 99 bp of covering sequence). However, this RAZA fragment is determined to lack the 5 'end of this gene. Therefore, a new RAZA 5 'PCR is carried out using gene-specific primers ManSRaza2 and ManSRazal. This produced approximately PCR fragment of 400 base pairs, which is cloned into vector pCR4-T0P0 and then sequenced. This obtained sequence (pVC3 CcManS Race2) was 340 bp long and covers the 5 'end of the CcManS Race 1 fragment (Figure 2A, showing 38 bp of coating sequence). The various clones, as shown in Figure 2A, allowed the generation of the DNA alignment shown in Figure 2B, which shows the coating sequences of these clones. This DNA sequence information is used to find the complete ORF for CcManS of coffee mannan synthase. From the ManS 5 'end sequence of freshly isolated coffee (CcManS Raza2), and the almost complete length coding sequence in the pcccs46wl6i 11 cDNA, two new primers (ManS-Am3 and ManS-Am2, Table 7) are designed , which were able to specifically amplify the complete ORF sequence of coffee mannan synthase using cDNA made from C. cam eph or ra (BP-409) RNA or Coffea a rabi ca (T2308) isolated from grain in the yellow development stage (Table 7). This PCR amplification experiment resulted in the generation of the cDNA sequences that are contained in the plasmids pVC4 (robust cDNA), pVC6 (robust cDNA), and pVC7 (Arabian cDNA), respectively (Figure 2B). Sequence analysis of the pVC4 insertion indicates that this cDNA was 1898 bp, and encodes a 530 amino acid polypeptide (estimated molecular weight of 61.29 kDa). Note: the DNA sequence of the insert in pVC4 is found to have a base change causing a stop codon in the ORF. As explained in the legend of Figure 2B, this base change is a PCR error and is not encoded by the corresponding genomic sequence. Sequence analysis of the insertions of pVC6 and pVC7 showed that these cDNA sequences were 1897 bp long and each had a full ORF of 1590 bp, encoding molecular weight estimated 530 amino acids of 61.3 kDa and 61.15 kDa, respectively .

Table 7. Sequences of primers used for the amplification of cDNA sequences encoding CcManS full-length protein sequences.

These protein sequences are then aligned with the biochemically characterized mannan guar synthase protein sequence (CtManS), as well as two of the most closely related sequences found in the GenBank database, the product of one of which it has not been characterized (ie, I. Trifold). The result of this alignment (Figure 4) shows that the sequence Co ffea ca n eph o ra ManS (CcManS; pVC6) shows 74.7%, 65.9% and 58.7% identity with the sequences of C. tetragonoloba, A. thaliana and I Tr if ida, respectively. The sequence of arabidopsis in this alignment is also called AtCSLA9 (arabidopsis cellulose synthase as gene # 9 of the protein family A) and the protein encoded by this gene has very recently shown to have mannan synthesis activity, and to a lesser degree activity of synthesis of glucomannan (Liepman, A., Wilkerson, C., Keegstra, K. 2005 Expression of cellulose synt hase- 1 i ke (Csl) genes in insect celias reveáis the CslA family members encode mannan synthases Proc. Nati. Acad Sci. 102, 2221-2226). The high levels of identity between the guar and coffee protein sequences strongly supports the argument that the sequences of CcManS and CaManS encode the protein responsible for mannan synthesis in the coffee bean. It is also observed that the ManS sequences of Coffea canephora (pVC6) and Coffea arabica '(pVC7) share 98.5% identity, and have only 12 nucleotide differences, which translate into a difference of 8 amino acids .. It may be that these differences delicate proteins in mannan synthase contribute to the difference in extraction rates generally known to exist between these two types of coffee. An alignment of the insertion DNA sequences of pVC4 (CcManS), Pvc6 (CcManS), and pVC7 (CaManS) is done with the MansS cDNA sequences of C. t e t ra gon ol oba (AAR23313) and A. th a 1 i a n a (CAB82941) using ClustalW. This DNA alignment showed that the coffee sequences were, as noted above, almost identical. In contrast, the C. tet ra gon or l oba sequence showed approximately 67% homology with the coffee and Aan synthase sequences. tha 1 i a n a showed approximately 55% homology with the coffee mannan synthase sequences (CAB82941). In addition, the identity regions were scattered on a regular basis by all the complete sequences and in this way the contiguous regions of not very long identity are between the coffee sequences and the guar and arabidopsis sequences. Example 4 Expression Analysis of CcManS To ensure that the CcManS gene encodes a family member similar to cellulose synthase (Csl) with mannan synthase activity, this gene is shown to express only in the tissue (s) which show a high level of synthesis of galactomannan and mannan. The expression of CcManS is studied in several tissues of arabic and robusta using quantitative RT-PCR. The results obtained clearly show that the mannan synthase is both highly and almost exclusively expressed in the grain of both robusta and arabic, with the grain T2308 of arabia appearing as having slightly higher levels of mannan synthase expression than the grain BP409 of robust This suggests that there may be higher levels of mannan synthase activity in Arabica grain versus robust grain, particularly later in grain development. This difference in activity would lead to higher levels and / or different structures of the mannan / galactomannan found in the Arabian grain. Such differences could explain, generally, the greater difficulty experienced in the extraction of solid material of roasted grain, or processed, Arabica against robust grain. The light expression or not of mannan synthase is detected using QRT-PCR in the stem, roots, leaves, pericarp and tissues of the T2308 or robusta BP409 Arabic flower. The small green sturdy sample was only the grain sample that does not have detectable mannan synthase gene expression, and this is in agreement with the previous results showing that this particular stage / sample of robusta does not yet express other specific genes of endosperm such as oleosins (see, for example, Co-pending Application, commonly owned No. 60 / 696,445). In sum, all mannan synthase expression data show that the mannan synthase is exclusively, or almost exclusively, expressed in the coffee bean in the later stages of development when the endosperm is forming or developing. Consistent with this discovery, ESTs of mammalian synthase were also only detected in libraries made of RNA extracted from grain in later stages of development, and not in libraries made of RNA extracted from early stage coffee cherries, pericarp tissues. of coffee cherry, or of leaf tissues (see Table 6). In summary, the data of mannan synthase expression are consistent with the theory that the mannan synthase gene encodes the main enzyme included in mannan synthesis, and by association, the main enzyme included in the galactomannan synthesis, in the coffee bean.

ND = not detected Table 8. Relative expression of CcManS vs. CcRpl39 Example 5 Identification of cDNA encoding (1, 6) -alpha-D-galactosyl transferase (GMGT) specific for mannan dependent on UDP-Gal A second enzyme included in the synthesis of galactomannans is the enzyme Mn ++ dependent, (1, 6) - alpha-D-Galactosyltransferase (GMGT) specific for mannan dependent on UDP-Gal (Edwards, Choo, Dickson, Scout, Gridley, and Reid 2004). GMGT along with mannan synthase is thought to work in close association, possibly as a complex to generate galactomannans. The protein sequence of one. GMGT protein biochemically characterized from Lo t us j apon i c u s (access number AJ567668) is used to search our set 'unigen' for DNA sequences using the tblastn algorithm (Altschul, et al., 1990). This search discovered two unigenes with a high level of homology (unite # 122567 and unigen # 122620). Table 9 shows the number of ESTs found for each unit in the different libraries of C. ca n eph ora. Since EST's of 122620 are only found in the seed, and that EST's for 122567 are only found in the leaf, it is likely that the # 122620 unite represents a gene that encodes a grain specific coffee GMGT. In the following, this GMGT protein (CcGMGTl) is thought to work with CcMans, described herein, to synthesize the vast majority of coffee bean galactomannans. In contrast, the gene represented by unigen # 122567 is likely to encode another GMGT protein from coffee (GMGT2), which is associated with galactomannan synthesis in other coffee tissues such as the leaf. The alignments of each unigen are shown in Figures 5 and 6. The CcGMGT1 ORF encoded by the unigen 122620 is found to have 54.3% identity with the fenugreek protein sequence and 53.6% identity with the Japanese protein sequence. what's The ORF of CcGMGT2 encoded by the unigen 122567 is found to have 62.8% identity with the fenugreek protein sequence and 63.8% identity with the protein sequence of Japon i cus. Equipped with these partial cDNA sequences, the full-length cDNA can be isolated for each gene using the well-established techniques of 5 'RACE and primer-assisted genome walking. Full-length cDNA for GMGT1 can be used to express a GMGT protein of active coffee bean in plant tissues such as coffee, and in overexpression model organisms, to generate proteins for functional analysis with the protein synthase of well The CcManS and CcGMGT proteins of coffee can be expressed at high levels in the same plant cell, yeast or bacterial, which could lead to the generation of substantial amounts of galactomannans produced by these cells of different type.

Table 9. Mass distribution of GMGT ESTs The number of ESTs GMGT found for each unit in the various libraries of Coffea ca n eph o ra is given Example 6 Isolation of a DNA sequence encoding the complete GMGTase 1 polypeptide sequence Example 5 presented the discovery of a partial cDNA sequence encoding (1, 6) -alpha-D-galact os i lt rans ferase specific to mannan depending on UDP-Gal, CcGMGTase 1 (CcGMTGl) C grain. ca n eph prays. To confirm the sequence of unigen # 122620 presented in Example 5, the second longest EST in the umgen (fish 46w8o23) is completely sequenced. To obtain the sequence data for CcGMGTasel upstream of the 5 'end of the fish partial cDNA sequence 46w8o23, RACE 5' is carried out with the GMGT-3 Owl 5mi 4 -RAZA 4 primers and GMGT-30wl5ml4-RAZA 2 (see Table 10 for the sequences). Using RNA isolated from the Arabian cherry grain T2308 in the "yellow" stage, cDNA is prepared as described above in the methods for this application. A poly dC backing is then added to the Arabian cDNA using the TdT enzyme and used in the 5 'RAZA reaction under the conditions described in the methods section. The first round of RAZA 5 'used the primers GMGT- 30wl 5ml 4 -RAZA 4 and AAP, and the second round of RAZA 5 'used the primers GMGT- 30 l 5mi 4 -RAZA 2 and AUAP. The tempering temperature in both reactions was 60 ° C. This produced a fragment of approximately 1.0-1.1 kilobase pair that is cloned into the pCR-4-TOPO vector and then sequenced.

Table 10. List of primers used for PCR RAZA 5 'experiments RACE 5 'generated the pVClO clone that contains an insert of 1120bp. The analysis of the complete sequence of this RAZA 5 'product showed that it encodes the N terminal region of GMGTase 1 of coffee. The complete ORF sequence of GMGTase 10 1 of coffee is successfully amplified by PCR as a single fragment of T-2308 genomic DNA of the arabian variety using a new set of PCR primers that is designed from the 5 'end of pVClO and the non-target region. 3 'coding of pcccs46w8o23 of cDNA. These oligonucleotides specific for GMGTase 1 GMGT-Fwdl and GMGT-Rev (Table 11) are then used to PCR amplify a fragment containing the complete ORG sequence of GMGTase 1 of the T-2308 arabic genomic DNA that has been purified from tissue of leaf according to the method previously described (Crouzillat et al., 1996 Theor, Appl. Genet, 93, 205-214). The PCR reaction is carried out in a 50 μl reaction as follows: 5 μl of gDNA, 5 μl 10 x PCR regulator (ThermoPol regulator), 400 nM of each gene-specific primer, 200 μM of each dNTP, and 0.5 U of polymerase of Taq DNA (Biolabs). After denaturing at 94 ° C for 2 min, the amplification consisted of 40 cycles of 1 min at 94 ° C, 1.5 minutes at 58 ° C, and 3 minitos at 72 ° C. An additional final stage of elongation is made at 72 ° C for 7 min. The PCR products are then analyzed by agarose gel electrophoresis and ethidium bromide staining. Fragments of the expected size (~ 1700 bp) are then cloned into pCR4-TOPO using the TOPO TA Cloning kit for Sequencing (Invitrogen) according to the instructions given by the manufacturer. The insertions of the generated plasmids are then completely sequenced. The sequence analysis of the obtained clone (pVCll, CaGMGTasel) showed that GMGTase 1 does not contain any intron in the majority of the coding sequence of this gene (intromens can still occur in the 5 'or 3' end coding regions of this gen).

Table 11. Sequences of the primers used for the amplification of a genomic sequence encoding the full-length protein sequence of Ca GMGTl The three clones used to obtain full-length coffee GMGTase 1 polypeptide sequence are presented in Figure 7. The generated DNA sequences are aligned using the CLUSTAL program (Figure 8). This alignment shows that there are some differences in the nucleic acid sequences obtained. However, only two of the base differences in the amino acid sequence region result in amino acid changes (position 432 has L against P and the position has 44 E against G). The complete amino acid sequence encoded by pVCll is then aligned with most of the homologous DNA sequences found in the GenBank public database. The result of this amino acid sequence alignment is shown in Figure 9. The CaGMGTase 1 sequence is most highly related to the galactomannan galactosyltransferase of Senna occi den ta l es (65% identity) and had approximately 56-57.6% identity with most of the other protein sequences in Figure 3, supporting the annotation of the full length polypeptide sequence of CaGMGTase 1 as a galactomannan galactosyltransferase. Example 7 Characterization of a cDNA encoding the complete GMGTase 2 polypeptide sequence Example 5 presented the discovery "of a partial cDNA sequence encoding (1, 6) -alta-D-galactos ilt rans ferase-specific mannan dependent on UDP-Gal, CcGMGTase 2 (CcGMGT2) of leaves of C. ca n eph o ra. This sequence of unigen (unite # 122567) is generated using three homologous EST sequences. To confirm the sequence data of the unigen, and to extend the sequence data to cover the 3 'end of the sequence, the longer EST clone in that set of unite was sequenced (clone pcccl26f9). The alignment of the complete DNA sequence of pcccl26f9 against the sequence of unigen # 122567 is present in Figure 10. As expected, the complete sequence of pcccl26f9 contained the 3 'end of CcGMGTase 2, as indicated by the presence of a poly A back. The ORF encoded by pcccl26f9 also contained the N-terminal region of GMGTase 2. The DNA sequence in the 5 'end of pcccl26f9 is almost identical to that of the unigen. However, a closer examination of the unigen sequence reveals that the first methionine codon of the pcccl26f9 (ATG) sequence was currently ATC in the unigenic sequence, thus the N-terminal amino acid sequence obtained from the sequence of DNA "unigen is not observed." The amino acid sequence encoded by pcccl26f9 is then aligned with several of the most closely related sequences found in the public GenBank database (Figure 11) .The examination of this alignment indicates that, although the GMGTase sequences 1 and GMGTase 2 of coffee have significant regions of homology (they have approximately 52% identity), are clearly encoded by different genes.This alignment also shows again that GMGTase 2 of coffee is also highly related to a group of proteins scored as galactosyltransferase. In conclusion, the evidence presented here strongly indicates that the cDNA clone isolated from the bibliotec a of coffee leaf EST (pcccl26f9) encodes the complete polypeptide sequence for GMGTase of coffee that is expressed in the coffee leaf. Example 8 Analysis of GMGTase 1 expression of coffee The expression levels of GMGTase 1 in various arabic and robusta tissues is analyzed using quantitative RT-PCR and the relative quantification approach (expression relative to rpl39). The method used was similar to that described earlier to measure the expression of coffee grain mannan synthase The specific primer and probe sets used are presented in Table 12. Amplification efficiency measurements of the primer set / TaqMan probe demonstrated that they were in the acceptable range of efficiency.CADN is prepared as described earlier in this application using SuperScript III (Invitrogen).

Table 12. Sequences of the primers and probes used for the quantitative RT-PCR experiments The results are presented in Figure 12 and demonstrate that GMGTase 1 is expressed mainly in the grain of both robusta and arabia. More and more interesting, there is a difference of about ten times in RQ found for the samples of arabic cDNA against robusta tested. It is possible that this difference in expression may contribute to some variation in either the level of galactoman and / or structure in the grain of the two species. It is also observed that the expression of GMGTase 1 in robusta is higher in the yellow stage. These contrasts with Arabic where the highest expression is observed in large green and small green stages. The expression GMGTase 1 is also detected at lower levels in most other tissues tested, again with the highest expression being detected in Arabic than robust. Finally, it is observed that the expression pattern observed for GMGTase 1 mimics the expression pattern observed for the expression of mannan synthase. Because these two proteins are proposed to work together in galactomannan synthesis, the GMGTase 1 expression data further support that GMGTase 1 is a key participant in the synthesis of coffee bean galactose. References: Altschul S.F., Madden T.L., Schaffer A.A., Zhang J., Zhang Z., Miller. and Lipman DJ. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein datbase search programs. Nu cl ei c Aci ds Res. 25: 3389-3402. Bacic A, Harris P, Stone B (1988) Structure and function of plant cell walls. In J Priess, ed, The biochemistry of plants; a comprehens i ve treatise, Vol 14 Carbohydrat en, Academic Press, New York, pp 297-371. Buckeridge M, Pessosa dos Santos H, Tine M (2000) Mobilization of storage cell wall polysacchar ides in seeds. Plant Physiol Biochem 38: 141-156. Charles-Bernard M, Kraehenbuehl K, Rytz A, Roberts D (2005) Interactions between volatile and non-volatile coffee components. 1. Screening of non-volatile components. J Agrie Food Chem 53: 4417-4425. Crouzillat D., Lerceteau E., Petiard V., Morera J., Rodriguez H., Waiker D., Philips W.R.R., Schnell J., Osei J. and Fritz P. (1996). Theobroma cacao L .: a genetic linkage map and quantitative trait loci analysis. Th eor Appl Gen e t. 93: 205-214. Cutler S, Somerville C (1997) Cloning in silico. Curr Biol 7: R108-R111. Dhugga KS, Barreiro R, Whitten B, Stecca K, Hazebroek J, Randhawa GS, Dolan M, Kinney AJ, Tomes D, Nichols S, Anderson P. (2004) Guar seed beta-mannan synthase is a member of the cellulose synthase super gene family. Sci in ce. 2004 Ja n 1 6; 303 (5656): 363-6. Edwards M, Choo T, Dickson C, Scott C, Gridley M, Reid J (2004) The seeds of Lotud japonicus lines. Translated with sense, antisense, and sense / ant i sense galacton omannan galactosyl trans ferase constructs have structurally 1 altered galactomannans in their endosperm cell walls. Plant Physiol 134: 1153-1162. Edwards M, Scott C, Gidley M, Reid J (1992) Control of mannose / galactose ratio during galact omannan formation in developing legume seeds. Floor 187: 67-74. Fischer M, Reimann S, Trovato V, Redgwell RJ (2001) Polysaccharides of green Arabica and Robusta coffee beans. Carbohydrate Research 330: 93-101. Fry S (2004) Primary cell wall metabolism: tracking the careers of wall polymers in living plant cells. New Phytologist 161: 641-675. Handford M, Baldwin T, Goubet F, Prime T, Miles J, Yu X, Dupree P (2003) Localization and charact eri zat ion of cell wall mannan polysacchar ised in Arabidopsis thaliana. Plant 218: 27-36. Hanford M, Baldwin T, Goubet F, Prime T, Miles J, Yu X, Dupree P (2003) Localisati? N and characteri zat ion of cell wall mannan polysaccharides in Arabidopsis thaliana. Plant 218: 27-36. Hazen SP, Scott-Craig JS, Walton JD (2002) Cellulose synt hase-li ke genes of rice. Plant Physiol 128: 336-340. Illy A, Viani R (1995) Expresso Coffee.

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Reid J, Bewley J (1979) A dual role for the endosperm and its galactomannan reserves in the germinative physiology of fenugreek (Tri gon el l a foen um -gra ec um L.) an endospermic leguminous seed. Floor 147: 145-150. Richmond TA, Somerville CR (2000) The cellulose synthase superfamily. Plant Physiol 124: 495-498. Schroder, R., Nicholas, P., Vincent, S., Fischer, M, Reymond, S., and Redgewell, R. (2001), Purification and character and zat ion of a galactoglucomannan from kiwi fruit (Actinidia deliciosa) Carbohydr Res. 331, 291-306. Sims, I. and Craik, D., and Bacic, A. (1997) Structural characteri zat ion of galactoglucomannan secreted by suspension-cul tured cells of Nicotiana plumbagini fol ia Carbohydr Res 303, 79-92. Somerville C, Bauer S, Brininstool G, Facette M, Hamann T, Milne J, Osborne E, Paredez A, Persson S, Raab T, Vorwerk S, Youngs H (2004a) Toward a systems approach to under standing plant cell walls. Science 306: 2206-2211. Somerville C, Bauer S, Brininstool G, Facette M, Hamann T, Milne J, Osborne E, Paredez A, Persson S, Raab T, Vorwerk S, Youngs H (2004b) Toward a systems approach to understanding plant cell walls. Science 306: 2206-2211. Somerville C, Bauer S, Brininstool G, Facette M, Hamann T, Milne J, Osborne E, Paredez A, Persson S, Raab T, Vorwerk S, Youngs H (2005) Toward a systems approach to understanding plant cell walls. Science 306: 2206-2211. Sunderland P, Hallet I, MacRea E, Fischer M, Redgwell R (2004) Cyt ochemis t ry and immunolocal i zat ion of polysaccharides and prot eoglycans in the endosperm of green Arabica coffee beans. Protoplasma 223: 203-211. Yeretzian C, Jordan A, Badoud R, Lindinger W (2005) From the green bean to the coffee cup: invest igating coffee roasting by on-line monitoring of volatiles. Eur Food Res Technol 214: 92-104. 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 (31)

1. Nucleic acid molecule isolated from Coffea spp. comprising a coding sequence encoding a galactomannan synthesis enzyme.

2. Nucleic acid molecule according to claim 1, wherein the galactomannan synthesis enzyme is a galactosyltransferase or a mannan synthase.

3. Nucleic acid molecule according to claim 2, wherein the mannan synthase comprises a conserved domain having amino acid sequence QHRWS.

4. Nucleic acid molecule according to claim 2, wherein the mannan synthase comprises an amino acid sequence greater than about 75% identical to that of any of SEQ ID NOS: 4-6. •

5. Nucleic acid molecule according to claim 2, wherein the mannan synthase comprises any of SEQ ID NOS: 4-6.

6. Nucleic acid molecule according to claim 2, comprising SEQ ID NO: 2 or SEQ ID NO: 3.

7. Nucleic acid molecule according to claim 2, wherein the galactosyltransferase has at least about 54% identity with a fenugreek galactosyltransferase or a galactosyltransferase of Lo t u s j apon i c u s.

8. Nucleic acid molecule according to claim 2, wherein the galactosyltransferase comprises an amino acid sequence greater than about 75% identical to any of SEQ ID NOS: 15-18.

9. Nucleic acid molecule according to claim 2, wherein the galactosyltransferase comprises any of SEQ ID NOS: 15-18.

10. Nucleic acid molecule according to claim 2, comprising any of SEQ ID NOS: 11-14.

11. Nucleic acid molecule according to claim 1, wherein the coding sequence is an open reading frame of a gene.

12. mRNA molecule produced by transcription of the gene according to claim 11.

13. cDNA molecule produced by reverse transcription of the mRNA molecule according to claim 12.

14. Oligonucleotide between 8 and 100 bases in length, which is complementary to a segment of the nucleic acid molecule according to claim 1.

15. Vector comprising the coding sequence of the nucleic acid molecule according to claim 1.

16. Vector according to claim 15, which is an expression vector selected from the group of vectors. which consists of plasmid, phagemid, cosmid, baculovirus, bacmido, bacterial, yeast and viral vectors.

17. Vector according to claim 15, wherein the coding sequence of the nucleic acid molecule is operably linked to a constitutive promoter.

18. A vector according to claim 15, wherein the coding sequence of the nucleic acid molecule is operably linked to an inducible promoter.

19. A vector according to claim 15, wherein the coding sequence of the nucleic acid molecule is operably linked to a tissue-specific promoter.

20. Vector according to claim 19, wherein the tissue-specific promoter is a seed-specific promoter.

21. Vector according to claim 20, wherein the seed-specific promoter is a specific promoter of coffee seed.

22. The host cell transformed with the vector according to claim 15.

23. The host cell according to claim 22, selected from the group consisting of plant cells, bacterial cells, fungal cells, insect cells and mammalian cells.

24. Host cell according to claim 23, which is a plant cell selected from the group of plants consisting of coffee, tobacco, Arabidopsis, corn, wheat, rice, soybean barley, rye, oats, sorghum, alfalfa, clover, canola , safflower, sunflower, peanut, cocoa, tomatillo, potato, pepper, eggplant, sugar beet, carrot, cucumber, lettuce, pea, aster, begonia, chrysanthemum, delphinium, petunia, zinnia, and herbs.

25. Fertile plant produced from the plant cell according to claim 24.

26. Method to modulate the capacity of extraction of solids from coffee beans, comprising modulating the production or activity of galactomannan synthesis enzyme within the coffee seeds.

27. The method of claim 26, wherein the galactomannan synthesis enzyme is a galactosyltransferase or a mannan synthase. The method according to claim 27, comprising increasing the production or activity of the galactosyltransferase, mannan synthase, or a combination thereof. 29. The method of claim 27, comprising increasing the expression of a gene encoding galactosyltransferase, mannan synthase, or a combination thereof within coffee seeds. 30. The method of claim 27, comprising introducing a transgene encoding galactosyltransferase, transgene encoding mannan synthase, or a combination thereof into the plant. The method according to claim 27, comprising decreasing the production or activity of the galactosyltransferase, mannan synthase, or a combination thereof.