NUCLEIC ACIDS AND PROTEINS ASSOCIATED WITH SUCROSE ACCUMULATION IN COFFEE

This claims benefit of U.S. Provisional Application No. 60/709,043 filed August 17, 2005, the entire contents of which are incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to the field of agricultural biotechnology. More particularly, the invention relates to enzymes participating in sucrose metabolism in plants, coffee in particular, and the genes and nucleic acid sequences that encodes these enzymes, along with regulatory mechanisms that regulate the sucrose metabolism via these enzymes.

BACKGROUND OF THE INVENTION

Various publications, including patents, published applications and scholarly articles, cited throughout the present specification are incorporated by reference herein, in their entireties. Citations not fully set forth within the specification may be found at the end of the specification.

Sucrose plays an important role in the ultimate aroma and flavor that is delivered by a coffee grain or bean. Sucrose is a major contributor to the total free reducing sugars in coffee, and reducing sugars are important flavor precursors in coffee. During the roasting of coffee grain, reducing sugars will react with amino group containing molecules in a Maillard type reaction, which generates a significant number of products with caramel, sweet and roast/burnt-type aromas and dark colors that are typically associated with coffee flavor (Russwurm, 1969; Holscher and Steinhart, 1995; Badoud, 2000). The highest quality Arabica grain (Coffea Arabica) have been found to have appreciably higher levels of sucrose (between 7.3 and 11.4 %) than the lowest quality Robusta grain (Coffea canephora) (between 4 and 5 %) (Russwurm, 1969; Illy and Viani, 1995; Chahan et al., 2002; Badoud, 2000). Despite being significantly degraded during roasting, sucrose still remains in the roasted grain

at concentrations of 0.4-2.8 % dry weight (DW); thereby, contributing directly to coffee sweetness. A clear correlation exists between the level of sucrose in the grain and coffee flavor. Therefore, identifying and isolating the major enzymes responsible for sucrose metabolism and the underlying genetic basis for variations in sucrose metabolism will enable advances in the art of improving coffee quality.

Currently, there are no published reports on the genes or enzymes involved in sucrose metabolism in coffee. However, sucrose metabolism has been studied in tomato Lycopersicon esculentum (a close relative of coffee, both are members of asterid I class), especially during tomato fruit development. An overview of the enzymes directly involved in sucrose metabolism in tomato is shown in Figure 1 (Nguyen-Quoc et al, 2001). The key reactions in this pathway are (1) the continuous rapid degradation of sucrose in the cytosol by sucrose synthase (SuSy) and cytoplasmic invertase (I), (2) sucrose synthesis by SuSy or sucrose-phosphate synthase (SPS), (3) sucrose hydrolysis in the vacuole or in the apoplast (region external to the plasma membrane, including cell walls, xylem vessels, etc) by acid invertase (vacuolar or cell wall bound) and, (4) the rapid synthesis and breakdown of starch in the amyloplast.

As in other sink organs, the pattern of sucrose unloading is not constant during tomato fruit development. At the early stages of fruit development, sucrose is unloaded intact from the phloem by the symplast pathway (direct connections between cells) and is not degraded to its composite hexoses during unloading. Both the expression and enzyme activity of SuSy are highest at this stage and are directly correlated with sucrose unloading capacity from the phloem (phenomena also called sink strength; Sun, et al., 1992; Zrenner et al., 1995). Later in fruit development, the symplastic connections are lost. Under these conditions of unloading, sucrose is rapidly hydrolyzed outside the fruit cells by the cell wall bound invertase and then the glucose and fructose products are imported into the cells by hexose transporters. Sucrose is subsequently synthesized de novo in the cytoplasm by SuSy or SPS (Figure 1). SPS catalyses an essentially irreversible reaction in vivo due to its close association with the enzyme sucrose phosphate phosphatase (Echeverria et al., 1997). In parallel to the loss of the symplastic connections, SuSy activity decreases, and eventually becomes undetectable in fruit at the onset of ripening (Robinson et al. 1998; Wang et al. 1993). Therefore, late in the development of tomato fruit, the SPS enzyme, in association with SP, appears as the major enzymes for sucrose synthesis.

During the past decade, evidence has increasingly indicated that SuSy is responsible for the cleavage of newly imported sucrose, thereby controlling the import capacity of the fruit (N'tchobo et al., 1999) and the rate of starch synthesis. At the same time, SPS is now considered a rate limiting enzyme in the pathway providing sucrose to plant storage organs (roots, tubers and seeds) commonly referred to as sink. Together, this growing body of data strongly indicates that SuSy and SPS enzymes are important regulators of sucrose metabolism during tomato fruit development.

Alterations in carbon partitioning in plants, and most particularly improvement of sucrose levels in sink organs, have already been successfully accomplished in several plants, the most extensive and most encouraging results being obtained in tomato (Lycopersicon esculentuni). Worrell and co workers have made a set of constructions to test the effects of increasing SPS levels. For the principle experiments, they used a maize SPS cDNA under the control of the SSU promoter (Rubisco small subunit promoter) (Worrell, et al., 1991; Galtier et al. 1993; Foyer and Ferrario, 1994; Micallef, et al., 1995; Van Assche et al., 1999; Nguyen-Quoc et al., 1999). The total SPS activity in the leaves of the transformed plants was six times greater than that of the controls, while the total SPS activity in the mature fruit from the transformed plants was only twice than that of untransformed controls. This observation suggests that, even with a strong constitutive promoter, the level of recombinant SPS was altered in a tissue specific manner. Interestingly, some results have also suggested that the maize SPS activity was not under circadian control when this enzyme was expressed in tomato (Galtier et al., 1993). It should also be noted that SPS enzyme activity is negatively regulated at the post-translational level by phosphorylation and the level of phosphorylation varies according to the level of light and thus the light and dark phases of photosynthesis (Sugden et al., 1999; Jones et Ort, 1997). Therefore, the latter result suggests that the increase of SPS activity in the transgenic plants was both due to an over-expression of the protein and to the unregulated activity of the transfected maize SPS enzyme {i.e., the regulation by phosphorylation was perturbed). The increase in SPS activity was accompanied by a significant increase (25%) in total overall SuSy activity in 20 day old tomato fruit. The SuSy activity was measured with an assay in the direction of sucrose breakdown (Nguyen- Quoc et al.; 1999). Fruit from these transgenic tomato lines showed higher sugar content (36 % increase) compared to untransformed plants (Van Assche et al., 1999). Biochemical studies have also shown that the high levels of the corn SPS activity in the plants caused a

modification of carbohydrate portioning in the tomato leaves with an increase of sucrose/starch ratios and also a strong improvement in photosynthetic capacity. The tomato plants appeared to tolerate the elevated levels of SPS as there were no apparent detrimental growing effects. Other plants transformed with the construct 35SCaMV-SPS (35 S Cauliflower Mosaic Virus) have three to five times more total SPS activity in leaves than in wild-type plants but surprisingly tomato fruit obtained from these particular transformants did not show any increase in SPS activity (Laporte et al. 1997; Nguyen-Quoc et al. 1999). These results indicate that the promoter selected to drive transgene expression could play an important role.

There remains a need to determine the metabolism, of sucrose in coffee and the enzymes involved in the metabolism. There is also a need to identify and isolate the genes that encode these enzymes in coffee, thereby providing genetic and biochemical tools for modifying sucrose production in coffee beans to manipulate the flavor and aroma of the coffee.

SUMMARY OF THE MVENTION

One aspect of the invention features a nucleic acid molecule isolated from coffee (Coffea spp.) comprising a coding sequence that encodes a sucrose synthase, sucrose phosphate synthase or sucrose phosphatase. In one embodiment, the coding sequence encodes a sucrose synthase having an amino acid sequence comprising at least one fragment of SEQ ID NO:8 comprising residues 7-554 or 565-727. In another embodiment, the sucrose synthase has an amino acid sequence greater than 89% identical to SEQ ID NO: 8, and preferably comprises SEQ ID NO:8. In other embodiments, the polynucleotide encoding the sucrose synthase has 90% or greater identity to the coding sequence set forth in SEQ ID NO: 1, and preferably comprises SEQ ID NO: 1.

In another embodiment, the coding sequence encodes a sucrose phosphate synthase having an amino acid sequence comprising at least one fragment of SEQ ID NO: 9 comprising residues 168-439 or 467-644. In another embodiment, the sucrose phosphate synthase has an amino acid sequence greater than 83% identical to SEQ ID NO:9, and preferably comprises SEQ ID NO:9. In other embodiments, the nucleic acid molecule encoding sucrose phosphate synthase has a coding sequence greater than 79% identical to the coding sequence set forth in SEQ ID NO: 2, and preferably comprises SEQ ID NO:2.

In another embodiment, the coding sequence encodes a sucrose phosphatase having an amino acid sequence comprising residues 1-408 of SEQ ID NO: 10. In another embodiment, the sucrose phosphatase has an amino acid sequence greater than 81% identical to SEQ ID NO: 10, and preferably comprises SEQ ED NO: 10. In another embodiment, the nucleic acid molecule comprises a coding sequence greater than 78% identical to the coding sequence set forth in SEQ ID NO:3, and preferably comprises SEQ ID NO:3.

In certain embodiments, the coding sequence of the nucleic acid molecule is an open reading frame of a gene. In other embodiments, it is a mRNA molecule produced by transcription of the gene, or a cDNA molecule produced by reverse transcription of the mRNA molecule.

Another aspect of the invention features an oligonucleotide between 8 and 100 bases in length, which is complementary to a segment of one of the aforementioned nucleic acid molecules.

Another aspect of the invention features a vector comprising the coding sequence of the nucleic acid molecule described above. In some embodiments, the vector is an expression vector selected from the group of vectors consisting of plasmid, phagemid, cosmid, baculovirus, bacmid, bacterial, yeast and viral vectors. In one embodiment, the coding sequence of the nucleic acid molecule is operably linked to a constitutive promoter. In another embodiment, the coding sequence of the nucleic acid molecule is operably linked to an inducible promoter. In another embodiment, the coding sequence of the nucleic acid molecule is operably linked to a tissue specific promoter, particularly a seed specific promoter, and more specifically a coffee seed specific promoter.

Another aspect of the invention features a host cell transformed with the vector described above. In various embodiments, the host cell is a plant cell, bacterial cell, fungal cell, insect cell or mammalian cell. In specific embodiments, the host cell is a plant cell from a plant such as coffee, tobacco, Arabidopsis, maize, wheat, rice, soybean barley, rye, oats, sorghum, alfalfa, clover, canola, safflower, sunflower, peanut, cacao, tomatillo, potato, pepper, eggplant, sugar beet, carrot, cucumber, lettuce, pea, aster, begonia, chrysanthemum, delphinium, zinnia, or turfgrasses.

In accordance with another aspect of the invention, a fertile plant produced from the aforementioned transformed plant cell is provided.

Yet another aspect of the invention provides a method of modulating flavor or aroma of coffee beans, comprising modulating production or activity of one or more sucrose metabolizing enzymes within coffee seeds. In one embodiment, the modulating comprises increasing production or activity of the one or more sucrose metabolizing enzymes. This may be accomplished increasing expression of one or more endogenous sucrose metabolizing enzyme-encoding genes within the coffee seeds, which, in certain embodiments, is achieved by introducing a sucrose metabolizing enzyme-encoding transgene into the plant. In one embodiment, the transgene encodes sucrose phosphate synthase. In a particular embodiment, the plant comprises more sucrose in its seeds than does an equivalent plant that does not contain the transgene. In another embodiment, the sucrose metabolizing enzyme is sucrose phosphate synthase and is modified by removal of one or more phosphorylation sites, thereby increasing activity of the enzyme.

In another embodiment, the method of modulation comprises decreasing production or activity of the one or more sucrose metabolizing enzymes. In certain embodiments, this may be accomplished by introducing a nucleic acid molecule into the coffee that inhibits the expression of one or more of the sucrose metabolizing enzyme-encoding genes.

Other features and advantages of the present invention will be understood by reference to the drawings, detailed description and examples that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Model for sucrose metabolism in tomato fruit. Sucrose (S) is imported from phloem by a symplastic pathway or is hydrolyzed by cell-wall invertase. Glucose and fructose are imported into the cytosol by specific Sugar Transporter Proteins. In cytosol, sucrose is degraded by sucrose synthase (SS) and its re-synthesis is catalysed by sucrose phosphate synthase(SPS) associated with sucrose phosphatase (SP) or SS. Sucrose can be exported in vacuole and hydrolysed by vacuolar invertase. UDP-glucose after modifications can be used for starch synthesis in chromoplast. Abbreviations: G, glucose; F, fructose; F6-P, fructose 6-phosphate; UDP-G, UDP-glucose; G6-P, glucose 6-phosphate; S6-P, sucrose 6- phosphate; I, invertase; SP, sucrose phosphatase; SPS sucrose phosphate synthase.

Figure 2. Protein sequence alignment of CcSS2 with other SuSy proteins sequences. CcSS2 protein is aligned with other sucrose synthase proteins available in the NCBI database was done using the CLUSTAL W program in the MegAlign software

(Lasergene package, DNASTAR). Amino acids underlined in red are different from CcSS2 protein. GenBank accession numbers are AY205084 for potato SuSyST2 {Solarium tuberosum) (SEQ ID NO.:11), AJ537575 for potato SuSyST4 (SEQ ID NO.:12)and AJOl 1535 for tomato SuSyLE2 (Lycopersicon esculentum) (SEQ ID NO.: 13).

Figure 3. Schematic represeutation of CcSPSl gene from C canephom. The SPS-Cl fragment has been amplified by PCR from BP-409 genomic DNA using the degenerate primers SPS-3 and SPS-4. Successive genome walking experiments subsequently permitted the amplification of the overlapping fragments for the 5' and 3' flanking regions of SPS-Cl. Alignment of the resulting genomic clones (Cl-12, C1-GW4-23, C 1-62 and Cl- GWl-11) has lead to the complete sequence of CcSPSl gene. The putative protein coding region has been localized by alignment with the closely related sucrose phosphate synthase protein SPSLEl (accession number No. AAC24872) (SEQ ID NO.: 14) from tomato {Lycopersicon esculentum). The protein-coding regions are shown in black. Triangles indicate the position of the translation initiation start (ATG) and stop (TAG) codons.

Figure 4. Protein sequence alignment of CcSPSl with other SPS proteins. Alignment of protein encoded by the CcSPSl cDNA with other SPS proteins available in the NCBI database was done using the CLUSTAL W program in the MegAlign software (Lasergene package, DNASTAR). Amino acids marked in red are different from the CcSPSl protein. The other SPS proteins, with the associated accession number in parentheses, are as follows: SPSST {Solarium tuberosum, CAA51872) (SEQ ID NO.: 15), SPSLEl {Lycopersicon esculentum, AAC24872) (SEQ ID NO.: 14), SPSNT {Nicotiana tabacum, AAF06792) (SEQ ID NO.: 16), SPSLE2 {Lycopersicon esculentum, AAU29197) (SEQ ID NO.:17). The three sites for potential seryl phosphorylation of CcSPS 1 protein are indicated by an asterisk (Serl50, Ser221 and Ser415). A highly conserved sequence surrounding each serine is also shown.

Figure 5. Protein sequence alignment of CcSPl with other SP proteins. Alignment of CcSPl protein with other SP proteins available in the NCBI database was done using the CLUSTAL W program in the MegAlign software (Lasergene package, DNASTAR). Amino acids underlined in red are different from CcSPl protein. GenBank accession numbers are NP_973609 for Arάbidopsis SPATl {Arάbidopsis thaliana) (SEQ ID NO.:18) and AAO33160 for tomato SPLE {Lycopersicon esculentum) (SEQ ID NO.:19).

Figure 6. Changes in activity of SPS and SuSy activity and concentrations of sucrose, glucose and fructose in whole grains (separated from pericarp and locules) during (A) FRT05 C. canephora and (B) CCCA12 C. arabica coffee grain maturation.

Coffee cherries at four different maturation stages characterized by size and color were used, i.e., SG (small green), LG (large green), Y (yellow) and R (red). Concentrations of sucrose, glucose and fructose in the coffee grain were measured in samples harvested in parallel to those used for the assays of SPS and SuSy activity. Sugar concentration is expressed in g / 100 g DW while enzymatic activities are expressed in μmoles/h/mg protein.

Figure 7. Tissue-specific mRNA expression profiles of CcSS2, CcSPSl and CcSPl in C. canephora (Robusta, BP409) and C. arabica (Arabica, T2308) using realtime RT-PCR. Total RNA was isolated from root, flower, leaf and coffee beans harvested at four different maturation stages i.e. Small-Green (SG), Large-Green (LG), Yellow (Y) and Red (R). For each maturation stage, coffee cherries have been separated from pericarp (P) and grains (G). Total RNA was reverse transcribed and subjected to real-time PCR using TaqMan-MGB probes. Relative amounts were calculated and normalized with respect to rpl39 transcript levels. Data shown represent mean values obtained from three amplification reactions and the error bars indicate the SD of the mean.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS Definitions

Various terms relating to the biological molecules and other aspects of the present invention are used through the specification and claims. The terms are presumed to have their customary meaning in the field of molecular biology and biochemistry unless they are specifically defined otherwise herein.

The term "sucrose metabolizing enzyme" refers to enzymes in plants that primarily function to accumulate sucrose or degrade sucrose within the plant and include, for example, sucrose synthase (SuSy), sucrose phosphate synthase (SPS) and sucrose phosphatase (SP). Together, the different sucrose metabolizing enzymes operate to control the metabolism of sucrose as needed by the plant for either storage or for energy needs.

"Isolated" means altered "by the hand of man" from the natural state. If a composition or substance occurs in nature, it has been "isolated" if it has been changed or

removed from its original environment, or both. For example, a polynucleotide or a polypeptie naturally present in a living plant or animal is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein.

"Polynucleotide", also referred to as "nucleic acid molecule", generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotides" include, without limitation single- and double- stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, "polynucleotide" refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other f reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. "Polynucleotide" also embraces relatively short polynucleotides, often referred to as oligonucleotides.

"Polypeptide" refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. "Polypeptide" refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. "Polypeptides" include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of

modifications. Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from natural posttranslational processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. See, for instance, Proteins - Structure and Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York, 1993 and Wold, F., Posttranslational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York, 1983; Seifter et al., "Analysis for Protein Modifications and Nonprotein Cofactors", Meth Enzymol (1990) 182:626-646 and Rattan et al., "Protein Synthesis: Posttranslational Modifications and Aging", Ann NY Acad Sci (1992) 663:48-62.

"Variant" as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties. A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, or deletions in any combination. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polynucleotide or polypeptide may be

naturally occurring, such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis.

In reference to mutant plants, the terms "null mutant" or "loss-of-function mutant" are used to designate an organism or genomic DNA sequence with a mutation that causes a gene product to be non-functional or largely absent. Such mutations may occur in the coding and/or regulatory regions of the gene, and may be changes of individual residues, or insertions or deletions of regions of nucleic acids. These mutations may also occur in the coding and/or regulatory regions of other genes which may regulate or control a gene and/or encoded protein, so as to cause the protein to be non-functional or largely absent.

The term "substantially the same" refers to nucleic acid or amino acid sequences having sequence variations that do not materially affect the nature of the protein (i.e. the structure, stability characteristics, substrate specificity and/or biological activity of the protein). With particular reference to nucleic acid sequences, the term "substantially the same" is intended to refer to the coding region and to conserved sequences governing expression, and refers primarily to degenerate codons encoding the same amino acid, or alternate codons encoding conservative substitute amino acids in the encoded polypeptide. With reference to amino acid sequences, the term "substantially the same" refers generally to conservative substitutions and/or variations in regions of the polypeptide not involved in determination of structure or function.

The terms "percent identical" and "percent similar" are also used herein in comparisons among amino acid and nucleic acid sequences. When referring to amino acid sequences, "identity" or "percent identical" refers to the percent of the amino acids of the subject amino acid sequence that have been matched to identical amino acids in the compared amino acid sequence by a sequence analysis program. "Percent similar" refers to the percent of the amino acids of the subject amino acid sequence that have been matched to identical or conserved amino acids. Conserved amino acids are those which differ in structure but are similar in physical properties such that the exchange of one for another would not appreciably change the tertiary structure of the resulting protein. Conservative substitutions are defined in Taylor (1986, J. Theor. Biol. 119:205). When referring to nucleic acid molecules, "percent identical" refers to the percent of the nucleotides of the subject nucleic acid sequence that have been matched to identical nucleotides by a sequence analysis program.

"Identity" and "similarity" can be readily calculated by known methods. Nucleic acid sequences and amino acid sequences can be compared using computer programs that align the similar sequences of the nucleic or amino acids and thus define the differences. In preferred methodologies, the BLAST programs (NCBI) and parameters used therein are employed, and the DNAstar system (Madison, WI) is used to align sequence fragments of genomic DNA sequences. However, equivalent alignments and similarity/identity assessments can be obtained through the use of any standard alignment software. For instance, the GCG Wisconsin Package version 9.1, available from the Genetics Computer Group in Madison, Wisconsin, and the default parameters used (gap creation penalty=12, gap extension penalty=4) by that program may also be used to compare sequence identity and similarity.

"Antibodies" as used herein includes polyclonal and monoclonal antibodies, chimeric, single chain, and humanized antibodies, as well as antibody fragments (e.g., Fab, Fab', F(ab')2 and Fy), including the products of a Fab or other immunoglobulin expression library. With respect to antibodies, the term, "immunologically specific" or "specific" refers to antibodies that bind to one or more epitopes of a protein of interest, but which do not substantially recognize and bind other molecules in a sample containing a mixed population of antigenic biological molecules. Screening assays to determine binding specificity of an antibody are well known and routinely practiced in the art. For a comprehensive discussion of such assays, see Harlow et al (Eds.), ANTIBODIES A LABORATORY MANUAL; Cold Spring Harbor Laboratory; Cold Spring Harbor, NY (1988), Chapter 6.

The term "substantially pure" refers to a preparation comprising at least 50-60% by weight the compound of interest (e.g., nucleic acid, oligonucleotide, protein, etc.). More preferably, the preparation comprises at least 75% by weight, and most preferably 90-99% by weight, the compound of interest. Purity is measured by methods appropriate for the compound of interest (e.g. chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like).

With respect to single-stranded nucleic acid molecules, the term "specifically hybridizing" refers to the association between two single-stranded nucleic acid molecules of sufficiently complementary sequence to permit such hybridization under pre-determined conditions generally used in the art (sometimes termed "substantially complementary"). In particular, the term refers to hybridization of an oligonucleotide with a substantially

complementary sequence contained within a single-stranded DNA or RNA molecule, to the substantial exclusion of hybridization of the oligonucleotide with single-stranded nucleic acids of non-complementary sequence.

A "coding sequence" or "coding region" refers to a nucleic acid molecule having sequence information necessary to produce a gene product, when the sequence is expressed. The coding sequence may comprise untranslated sequences {e.g., introns) within translated regions, or may lack such intervening untranslated sequences {e.g., as in cDNA).

"Intron" refers to polynucleotide sequences in a nucleic acid that do not code information related to protein synthesis. Such sequences are transcribed into mRNA, but are removed before translation of the mRNA into a protein.

The term "operably linked" or "operably inserted" means that the regulatory sequences necessary for expression of the coding sequence are placed in a nucleic acid molecule in the appropriate positions relative to the coding sequence so as to enable expression of the coding sequence. By way of example, a promoter is operably linked with a coding sequence when the promoter is capable of controlling the transcription or expression of that coding sequence. Coding sequences can be operably linked to promoters or regulatory sequences in a sense or antisense orientation. The term "operably linked" is sometimes applied to the arrangement of other transcription control elements (e.g. enhancers) in an expression vector.

Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.

The terms "promoter", "promoter region" or "promoter sequence" refer generally to transcriptional regulatory regions of a gene, which may be found at the 5 ' or 3' side of the coding region, or within the coding region, or within introns. Typically, a promoter is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3 ¹ direction) coding sequence. The typical 5' promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence is a transcription initiation site (conveniently defined by mapping with nuclease Sl), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.

A "vector" is a replicon, such as plasmid, phage, cosmid, or virus to which another nucleic acid segment may be operably inserted so as to bring about the replication or expression of the segment.

The term "nucleic acid construct" or "DNA construct" is sometimes used to refer to a coding sequence or sequences operably linked to appropriate regulatory sequences and inserted into a vector for transforming a cell. This term may be used interchangeably with the term "transforming DNA" or "transgene". Such a nucleic acid construct may contain a coding sequence for a gene product of interest, along with a selectable marker gene and/or a reporter gene.

A "marker gene" or "selectable marker gene" is a gene whose encoded gene product confers a feature that enables a cell containing the gene to be selected from among cells not containing the gene. Vectors used for genetic engineering typically contain one or more selectable marker genes. Types of selectable marker genes include (1) antibiotic resistance genes, (2) herbicide tolerance or resistance genes, and (3) metabolic or auxotrophic marker genes that enable transformed cells to synthesize an essential component, usually an amino acid, which the cells cannot otherwise produce.

A "reporter gene" is also a type of marker gene. It typically encodes a gene product that is assayable or detectable by standard laboratory means (e.g., enzymatic activity, fluorescence).

The term "express," "expressed," or "expression" of a gene refers to the biosynthesis of a gene product. The process involves transcription of the gene into mRNA and then translation of the mRNA into one or more polypeptides, and encompasses all naturally occurring post-translational modifications.

"Endogenous" refers to any constituent, for example, a gene or nucleic acid, or polypeptide, that can be found naturally within the specified organism.

A "heterologous" region of a nucleic acid construct is an identifiable segment (or segments) of the nucleic acid molecule within a larger molecule that is not found in association with the larger molecule in nature. Thus, when the heterologous region comprises a gene, the gene will usually be flanked by DNA that does not flank the genomic DNA in the genome of the source organism. In another example, a heterologous region is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the

native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein. The term "DNA construct", as defined above, is also used to refer to a heterologous region, particularly one constructed for use in transformation of a cell.

A cell has been "transformed" or "transfected" by exogenous or heterologous DNA when such DNA has been introduced inside the cell. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA. A "clone" is a population of cells derived from a single cell or common ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations.

"Grain," "seed," or "bean," refers to a flowering plant's unit of reproduction, capable of developing into another such plant. As used herein, especially with respect to coffee plants, the terms are used synonymously and interchangeably.

As used herein, the term "plant" includes reference to whole plants, plant organs (e.g., leaves, stems, shoots, roots), seeds, pollen, plant cells, plant cell organelles, and progeny thereof. Parts of transgenic plants are to be understood within the scope of the invention to comprise, for example, plant cells, protoplasts, tissues, callus, embryos as well as flowers, stems, seeds, pollen, fruits, leaves, or roots originating in transgenic plants or their progeny.

Description:

Sucrose is a major contributor of free reducing sugars involved in the Maillard reaction that occurs during the roasting of coffee grain. Therefore, it is widely believed to be an important flavor precursor molecule in the green coffee grain. Consistent with this idea, the highest quality Arabica grains have appreciably higher levels of sucrose (between 7.3 and 11.4 %) than the lowest quality Robusta grains (between 4 and 5 %). Also, sucrose, while being significantly degraded during roasting, can remain in the roasted grain at concentrations of 0.4-2.8 % dry weight (DW) and so participates directly in coffee's sweetness. Because of

the clear correlation between the level of sucrose in the grain and coffee flavor, the ability to understand and manipulate the underlying genetic basis for variations in sucrose metabolism and carbon partitioning in coffee grain is important.

Key enzymes involved in sucrose metabolism have been characterized in model organisms (e.g., tomato, potato, Arabidopsis). In accordance with the present invention, protein sequences of these enzymes have been used to perform similarity searches in Coffea canephora cDNA libraries and EST databases using the tBLASTn algorithm, as described in greater detail in the examples. cDNA encoding sucrose synthase (CcSS2) (SEQ ID NO:1), sucrose phosphate synthase (CcSPSl) (SEQ ID NO:2), sucrose phosphatase (CcSPl) (SEQ ID NO: 3) were identified and characterized in C. canephora. A partial cDNA sequence of CcSPSl has also been identified, and is referred to herein as SEQ ID NO: 7.

Using degenerate primers, a partial genomic clone of a sucrose phosphate synthase CcSPSl -encoding gene (SEQ ID NO:4) has been isolated from C. canephora. A second gene was also isolated, and referred to herein as CcSPS2 (SEQ ID NO: 5). Confirmation of expression was performed with CcSPSl by sequencing the single PCR fragment obtained after RT-PCR. A complete genomic clone of a CcSPSl -encoding gene was identified and is referred to herein as SEQ ID NO: 6.

Eleven single nucleotide polymorphisms (SNPs) have been identified in the CcSPSl full length genomic clone. It is expected that these SNPs and other sequence markers will be useful for placing the CcSPSl gene on a C. canephora genetic map.

The study of SuSy and SPS activity during grain development in a variety of Arabica (C Arabica CCCAl 2) and Robusta (C. canephora FRT05) grain has shown that, although the Robusta variety was characterized by a stronger sink strength (correlated to higher SuSy activity), the Arabica variety accumulated 30 % more sucrose in mature beans than did the Robusta variety. Additionally, while SPS activity fluctuated during the Robusta grain development, the SPS activity in Arabica rose rapidly and remained high up to grain maturity. It was found that CcSS2 and CcSPSl mRNA accumulation was highly correlated with enzymatic activity. The data obtained in accordance with the invention described herein strongly indicate that SPS activity is the limiting step for re-synthesis of sucrose during final step of coffee grain maturation. In the perspective of improving the quality of Robusta and other coffee grain, selection of varieties with high SPS activity, or manipulation of plants to

increase SPS production or activity, are expected to be an important route for increasing the final sucrose concentration in mature coffee bean.

Thus, one aspect of the present invention relates to nucleic acid molecules from coffee that encode a number of sucrose metabolizing enzymes, including sucrose synthase (SuSy), exemplified by SEQ ID NO:1, sucrose phosphate synthase (SPS), exemplified by SEQ ID NO:2 (and the partial sequence of SEQ ID NO:7), and the open reading frame of SEQ ID NO:6 (and by the partial open reading frames of SEQ ID NOS: 4 and 5 described herein), and sucrose phosphatase (SP), exemplified by SEQ ID NO:3. Other aspects of the invention relate to the proteins produced by expression of these nucleic acid molecules and their uses. The deduced amino acid sequences of the proteins produced by expression of SEQ ID NOS: 1, 2 or 3 are set forth herein as SEQ NO: 8 (SuSy), SEQ ID NO: 9 (SPS) and SEQ ID NO: 10 (SP). The predicted molecular masses of these proteins are 92.6 kDa (SuSy), 117 kDa (SPS) and 46.7 kDa (SP). Still other aspects of the invention relate to uses of the nucleic acid molecules and encoded polypeptides in plant breeding and in genetic manipulation of plants, and ultimately in the manipulation of coffee flavor, aroma and other qualities.

Although polynucleotides encoding sucrose metabolizing enzymes from Coffea canephora are described and exemplified herein, this invention is intended to encompass nucleic acids and encoded proteins from other Coffea species that are sufficiently similar to be used interchangeably with the C. canephora polynucleotides and proteins for the purposes described below. Accordingly, when the terms "sucrose synthase," "sucrose phosphate synthase," and "sucrose phosphatase" are used herein, they are intended to encompass all Coffea sucrose synthases, sucrose phosphate synthases, and sucrose phosphatases having the general physical, biochemical and functional features described herein, and polynucleotides encoding them.

Considered in terms of their sequences, sucrose metabolizing enzyme-encoding polynucleotides of the invention include allelic variants and natural mutants of any of SEQ ID NOS: 1-7, which are likely to be found in different varieties of C. canephora, and homologs of SEQ ID NOS: 1-7 likely to be found in different coffee species. Because such variants and homologs are expected to possess certain differences in nucleotide and amino acid sequence, this invention provides isolated sucrose metabolizing enzyme-encoding nucleic acid molecules that encode respective polypeptides having at least about 80% (and, with increasing order of preference, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,

91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99%) identity with the coding regions of any one of SEQ ID NOS: 8-10 and comprises a nucleotide sequence having equivalent ranges of identity to the pertinent portions of any one of SEQ ID NOS: 1-7, respectively. Because of the natural sequence variation likely to exist among sucrose metabolizing enzymes, and the genes encoding them in different coffee varieties and species, one skilled in the art would expect to find this level of variation, while still maintaining the unique properties of the polypeptides and polynucleotides of the present invention. Such an expectation is due in part to the degeneracy of the genetic code, as well as to the known evolutionary success of conservative amino acid sequence variations, which do not appreciably alter the nature of the encoded protein. Accordingly, such variants and homologs are considered substantially the same as one another and are included within the scope of the present invention.

The following sections set forth the general procedures involved in practicing the present invention. To the extent that specific materials are mentioned, it is merely for the purpose of illustration, and is not intended to limit the invention. Unless otherwise specified, general biochemical and molecular biological procedures, such as those set forth in Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory (1989) or Ausubel et al. (eds), Current Protocols in Molecular Biology, John Wiley & Sons (2005) are used.

Nucleic Acid Molecules, Proteins and Antibodies:

Nucleic acid molecules of the invention may be prepared by two general methods: (1) they may be synthesized from appropriate nucleotide triphosphates, or (2) they may be isolated from biological sources. Both methods utilize protocols well known in the art.

The availability of nucleotide sequence information, such as the cDNA having SEQ ID NOS: 1, 2 or 3, (or the open reading frame of SEQ ID NO: 6) enables preparation of an isolated nucleic acid molecule of the invention by oligonucleotide synthesis. Synthetic oligonucleotides may be prepared by the phosphoramidite method employed in the Applied Biosystems 38A DNA Synthesizer or similar devices. The resultant construct may be purified according to methods known in the art, such as high performance liquid chromatography (HPLC). Long, double-stranded polynucleotides, such as a DNA molecule of the present invention, must be synthesized in stages, due to the size limitations inherent in current oligonucleotide synthetic methods. Thus, for example, a long double-stranded molecule may be synthesized as several smaller segments of appropriate complementarity.

Complementary segments thus produced may be annealed such that each segment possesses appropriate cohesive termini for attachment of an adjacent segment. Adjacent segments may be ligated by annealing cohesive termini in the presence of DNA ligase to construct an entire long double-stranded molecule. A synthetic DNA molecule so constructed may then be cloned and amplified in an appropriate vector.

In accordance with the present invention, nucleic acids having the appropriate level sequence homology with part or all of the coding and/or regulatory regions of sucrose metabolizing enzyme-encoding polynucleotides may be identified by using hybridization and washing conditions of appropriate stringency. It will be appreciated by those skilled in the art that the aforementioned strategy, when applied to genomic sequences, will, in addition to enabling isolation of sucrose metabolizing enzyme-coding sequences, also enable isolation of promoters and other gene regulatory sequences associated with sucrose metabolizing enzyme genes, even though the regulatory sequences themselves may not share sufficient homology to enable suitable hybridization.

As a typical illustration, hybridizations may be performed, according to the method of Sambrook et al., using a hybridization solution comprising: 5X SSC, 5X Denhardt's reagent, 1.0% SDS, 100 μg/ml denatured, fragmented salmon sperm DNA, 0.05% sodium pyrophosphate and up to 50% formamide. Hybridization is carried out at 37-42°C for at least six hours. Following hybridization, filters are washed as follows: (1) 5 minutes at room temperature in 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 ⁰ C in 2X SSC and 0.1% SDS, changing the solution every 30 minutes.

One common formula for calculating the stringency conditions required to achieve hybridization between nucleic acid molecules of a specified sequence homology (Sambrook et al., 1989):

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

As an illustration of the above formula, using [Na+] = [0.368] and 50% formamide, with GC content of 42% and an average probe size of 200 bases, the Tm is 57 ⁰ C. The Tm of a DNA duplex decreases by 1 - 1.5 ⁰ C with every 1% decrease in homology. Thus, targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42°C. In one embodiment, the hybridization is at 37°C and the final wash is at

42°C; in another embodiment the hybridization is at 42°C and the final wash is at 50 ⁰ C; and in yet another embodiment the hybridization is at 42 ⁰ C and final wash is at 65°C, with the above hybridization and wash solutions. Conditions of high stringency include hybridization at 42°C in the above hybridization solution and a final wash at 65°C in 0.1X SSC and 0.1% SDS for 10 minutes.

Nucleic acids of the present invention may be maintained as DNA in any convenient cloning vector. In a preferred embodiment, clones are maintained in plasmid cloning/expression vector, such as pGEM-T (Promega Biotech, Madison, WI), pBluescript (Stratagene, La Jolla, CA), pCR4-TOPO (Invitrogen, Carlsbad, CA) or pET28a+ (Novagen, Madison, WI), all of which can be propagated in a suitable E. coli host cell.

Nucleic acid molecules of the invention include cDNA, genomic DNA, RNA, and fragments thereof which may be single-, double-, or even triple-stranded. Thus, this invention provides oligonucleotides (sense or antisense strands of DNA or RNA) having sequences capable of hybridizing with at least one sequence of a nucleic acid molecule of the present invention. Such oligonucleotides are useful as probes for detecting sucrose metabolizing enzyme encoding genes or mRNA in test samples of plant tissue, e.g., by PCR amplification, or for the positive or negative regulation of expression of sucrose metabolizing enzyme-encoding genes at or before translation of the mRNA into proteins. Methods in which sucrose metabolizing enzyme-encoding oligonucleotides or polynucleotides may be utilized as probes for such assays include, but are not limited to: (1) in situ hybridization; (2) Southern hybridization (3) northern hybridization; and (4) assorted amplification reactions such as polymerase chain reactions (PCR, including RT-PCR) and ligase chain reaction (LCR).

Polypeptides encoded by nucleic acids of the invention may be prepared in a variety of ways, according to known methods. If produced in situ the polypeptides may be purified from appropriate sources, e.g., seeds, pericarps, or other plant parts.

Alternatively, the availability of isolated nucleic acid molecules encoding the SuSy, SPS or SP polypeptides enables production of the proteins using in vitro expression methods known in the art. For example, a cDNA or gene may be cloned into an appropriate in vitro transcription vector, such as pSP64 or pSP65 for in vitro transcription, followed by cell-free translation in a suitable cell-free translation system, such as wheat germ or rabbit

reticulocytes. In vitro transcription and translation systems are commercially available, e.g., from Promega Biotech, Madison, WI, BRL, Rockville, MD or Invitrogen, Carlsbad, CA.

According to a preferred embodiment, larger quantities of sucrose metabolizing enzymes may be produced by expression in a suitable procaryotic or eucaryotic system. For example, part or all of a DNA molecule, such as the cDNAs having SEQ ID NOS: 1, 2 or 3, may be inserted into a plasmid vector adapted for expression in a bacterial cell (such as E. coli) or a yeast cell (such as Saccharomyces cerevisiae), or into a baculovirus vector for expression in an insect cell. Such vectors comprise the regulatory elements necessary for expression of the DNA in the host cell, positioned in such a manner as to permit expression of the DNA in the host cell. Such regulatory elements required for expression include promoter sequences, transcription initiation sequences and, optionally, enhancer sequences.

The sucrose metabolizing enzymes produced by gene expression in a recombinant procaryotic or eucyarotic system may be purified according to methods known in the art. In a preferred embodiment, a commercially available expression/secretion system can be used, whereby the recombinant protein is expressed and thereafter secreted from the host cell, and, thereafter, purified from the surrounding medium. An alternative approach involves purifying the recombinant protein by affinity separation, e.g., via immunological interaction with antibodies that bind specifically to the recombinant protein.

The sucrose metabolizing enzymes of the invention, prepared by the aforementioned methods, may be analyzed according to standard procedures.

Sucrose metabolizing enzymes purified from coffee or recombinantly produced, may 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 entire recombinant protein, if analyses of the proteins or Southern and cloning analyses (see below) indicate that the cloned genes belongs to a multigene family, then member-specific antibodies made to synthetic peptides corresponding to nonconserved regions of the protein can be generated.

Kits comprising an antibody of the invention for any of the purposes described herein are also included within the scope of the invention. In general, such a kit includes a control antigen for which the antibody is immunospecific.

Vectors, Cells, Tissues and Plants:

Also featured in accordance with the present invention are vectors and kits for producing transgenic host cells that contain a sucrose metabolizing enzyme-encoding polynucleotide or oligonucleotide, or homolog, analog or variant thereof in a sense or antisense orientation, or reporter gene and other constructs under control of sucrose metabolizing enzyme-promoters and other regulatory sequences. Suitable host cells include, but are not limited to, plant cells, bacterial cells, yeast and other fungal cells, insect cells and mammalian cells. Vectors for transforming a wide variety of these host cells are well known to those of skill in the art. They include, but are not limited to, plasmids, cosmids, baculoviruses, bacmids, bacterial artificial chromosomes (BACs), yeast artificial , chromosomes (YACs), as well as other bacterial, yeast and viral vectors. Typically, kits for producing transgenic host cells will contain one or more appropriate vectors and instructions for producing the transgenic cells using the vector. Kits may further include one or more additional components, such as culture media for culturing the cells, reagents for performing transformation of the cells and reagents for testing the transgenic cells for gene expression, to name a few.

The present invention includes transgenic plants comprising one or more copies of a sucrose metabolizing enzyme-encoding gene, or nucleic acid sequences that inhibit the production or function of a plant's endogenous sucrose metabolizing enzymes. This is accomplished by transforming plant cells with a transgene that comprises part of all of a sucrose metabolizing enzyme coding sequence, or mutant, antisense or variant thereof, including RNA, controlled by either native or recombinant regulatory sequences, as described below. Transgenic plants coffee species are preferred, including, without limitation, C. abeokutae, C. arabica, C. arnoldiana, C. aruwemiensis, C. bengalensis, C. canephora, C. congensis C. dewevrei, C. excelsa, C. eugenioides ,and C. heterocalyx, C. kapakata, C. khasiana, C. liberica, C. moloundou, C. rasemosa, C. sahatrix, C.sessiflora, C. stenophylla, C. travencorensis, C. wightiana and C. zanguebariae. Plants of any species are also included in the invention; these include, but are not limited to, tobacco, Arabidopsis and other "laboratory-friendly" species, cereal crops such as maize, wheat, rice, soybean barley, rye, oats, sorghum, alfalfa, clover and the like, oil-producing plants such as canola, safflower, sunflower, peanut, cacao and the like, vegetable crops such as tomato tomatillo, potato, pepper, eggplant, sugar beet, carrot, cucumber, lettuce, pea and the like, horticultural plants

such as aster, begonia, chrysanthemum, delphinium, petunia, zinnia, lawn and turfgrasses and the like.

Transgenic plants can be generated using standard plant transformation methods known to those skilled in the art. These include, but are not limited to, Agrobacterium vectors, polyethylene glycol treatment of protoplasts, biolistic DNA delivery, UV laser microbeam, gemini virus vectors or other plant viral vectors, calcium phosphate treatment of protoplasts, electroporation of isolated protoplasts, agitation of cell suspensions in solution with microbeads coated with the transforming DNA, agitation of cell suspension in solution with silicon fibers coated with transforming DNA, direct DNA uptake, liposome-mediated DNA uptake, and the like. Such methods have been published in the art. See, e.g., Methods for Plant Molecular Biology (Weissbach & Weissbach, eds., 1988); Methods in Plant Molecular Biology (Schuler & Zielinski, eds., 1989); Plant Molecular Biology Manual (Gelvin, Schilperoort, Verma, eds., 1993); and Methods in Plant Molecular Biology - A Laboratory Manual (Maliga, Klessig, Cashmore, Gruissem & Varner, eds., 1994).

The method of transformation depends upon the plant to be transformed. Agrobacterium vectors are often used to transform dicot species. Agrobacterium binary vectors include, but are not limited to, BINl 9 and derivatives thereof, the pBI vector series, and binary vectors pGA482, pGA492, pLH7000 (GenBank Accession AY234330) and any suitable one of the pC AMBIA vectors (derived from the pPZP vectors constructed by Hajdukiewicz, Svab & Maliga, (1994) Plant MoI Biol 25: 989-994, available from GAMBIA, GPO Box 3200, Canberra ACT 2601, Australia or via the worldwide web at CAMBlA.org). For transformation of monocot species, biolistic bombardment with particles coated with transforming DNA and silicon fibers coated with transforming DNA are often useful for nuclear transformation. Alternatively, Agrobacterium "superbinary" vectors have been used successfully for the transformation of rice, maize and various other monocot species.

DNA constructs for transforming a selected plant comprise a coding sequence of interest operably linked to appropriate 5' regulatory sequences {e.g., promoters and translational regulatory sequences) and 3' regulatory sequences {e.g., terminators). In a preferred embodiment, a sucrose metabolizing enzyme-encoding sequence under control of its natural 5' and 3' regulatory elements is utilized. In other embodiments, sucrose metabolizing enzyme-encoding and regulatory sequences are swapped to alter the sugar

profile of the transformed plant for a phenotypic improvement, e.g., in flavor, aroma or other feature.

In an alternative embodiment, the coding region of the gene is placed under a powerful constitutive promoter, such as the Cauliflower Mosaic Virus (CaMV) 35S promoter or the figwort mosaic virus 35S promoter. Other constitutive promoters contemplated for use in the present invention include, but are not limited to: T-DNA mannopine synthetase, nopaline synthase and octopine synthase promoters. In other embodiments, a strong monocot promoter is used, for example, the maize ubiquitin promoter, the rice actin promoter or the rice tubulin promoter (Jeon et al., Plant Physiology. 123: 1005-14, 2000).

Transgenic plants expressing sucrose metabolizing enzyme-coding sequences under an inducible promoter are also contemplated to be within the scope of the present invention. Inducible plant promoters include the tetracycline repressor/operator controlled promoter, the heat shock gene promoters, stress (e.g., wounding)-induced promoters, defense responsive gene promoters (e.g. phenylalanine ammonia lyase genes), wound induced gene promoters (e.g. hydroxyproline rich cell wall protein genes), chemically-inducible gene promoters (e.g., nitrate reductase genes, glucanase genes, chitinase genes, etc.) and dark-inducible gene promoters (e.g., asparagine synthetase gene) to name a few.

Tissue specific and development-specific promoters are also contemplated for use in the present invention. Non-limiting examples of seed-specific promoters include Ciml (cytokinin-induced message), cZ19Bl (maize 19 kDa zein), milps (myo-inositol-1 -phosphate synthase), and celA (cellulose synthase) (U.S. application Ser. No. 09/377,648), bean beta- phaseolin, napin, beta-conglycinin, soybean lectin, cruciferin, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein, waxy, shrunken 1, shrunken 2, and globulin 1, soybean HS legumin (Baumlein et al., 1992), and C. canephora HS seed storage protein (Marraccini et al, 1999, Plant Physiol. Biochem. 37: 273-282). See also WO 00/12733, where seed- preferred promoters from endl and end2 genes are disclosed. Other Coffea seed specific promoters may also be utilized, including but not limited to the oleosin gene promoter described in commonly-owned, co-pending Provisional Application No. 60/696,445 and the dehyrdin gene promoter described in commonly-owned, co-pending Provisional Application No. 60/696,890. Examples of other tissue-specific promoters include, but are not limited to: the ribulose bisphosphate carboxylase (RuBisCo) small subunit gene promoters (e.g., the coffee small subunit promoter as described by Marraccini et al., 2003) or chlorophyll a/b

binding protein (CAB) gene promoters for expression in photosynthetic tissue; and the root- specific glutamine synthetase gene promoters where expression in roots is desired.

The coding region is also operably linked to an appropriate 3' regulatory sequence. In embodiments where the native 3' regulatory sequence is not use, the nopaline synthetase polyadenylation region may be used. Other useful 3' regulatory regions include, but are not limited to the octopine synthase polyadenylation region.

The selected coding region, under control of appropriate regulatory elements, is operably linked to a nuclear drug resistance marker, such as kanamycin resistance. Other useful selectable marker systems include genes that confer antibiotic or herbicide resistances (e.g., resistance to hygromycin, sulfonylurea, phosphinothricin, or glyphosate) or genes conferring selective growth (e.g., phosphomannose isomerase, enabling growth of plant cells on mannose). Selectable marker genes include, without limitation, genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO), dihydrofolate reductase (DHFR) and hygromycin phosphotransferase (HPT), as well as genes that confer resistance to herbicidal compounds, such as glyphosate-resistant EPSPS and/or glyphosate oxidoreducatase (GOX), Bromoxynil nitrilase (BXN) for resistance to bromoxynil, AHAS genes for resistance to imidazolinones, sulfonylurea resistance genes, and 2,4- dichlorophenoxyacetate (2,4-D) resistance genes.

In certain embodiments, promoters and other expression regulatory sequences encompassed by the present invention are operably linked to reporter genes. Reporter genes contemplated for use in the invention include, but are not limited to, genes encoding green fluorescent protein (GFP), red fluorescent protein (DsRed), Cyan Fluorescent Protein (CFP), Yellow Fluorescent Protein (YFP), Cerianthus Orange Fluorescent Protein (cOFP), alkaline phosphatase (AP), β-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo ^r , G418 ¹ ) dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), lacZ (encoding α-galactosidase), and xanthine guanine phosphoribosyltransferase (XGPRT), Beta- Glucuronidase (gus), Placental Alkaline Phosphatase (PLAP), Secreted Embryonic Alkaline Phosphatase (SEAP), or Firefly or Bacterial Luciferase (LUC). As with many of the standard procedures associated with the practice of the invention, skilled artisans will be aware of additional sequences that can serve the function of a marker or reporter.

Additional sequence modifications are known in the art to enhance gene expression in a cellular host. These modifications include elimination of sequences encoding superfluous polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well-characterized sequences that may be deleterious to gene expression. Alternatively, if necessary, the G/C content of the coding sequence may be adjusted to levels average for a given coffee plant cell host, as calculated by reference to known genes expressed in a coffee plant cell. Also, when possible, the coding sequence is modified to avoid predicted hairpin secondary mRNA structures. Another alternative to enhance gene expression is to use 5' leader sequences. Translation leader sequences are well known in the art, and include the cis- acting derivative (omega ¹ ) of the 5' leader sequence (omega) of the tobacco mosaic virus, the 5' leader sequences from brome mosaic virus, alfalfa mosaic virus, and turnip yellow mosaic virus.

Plants are transformed and thereafter screened for one or more properties, including the presence of the transgene product, the transgene-encoding mRNA, or an altered phenotype associated with expression of the transgene. It should be recognized that the amount of expression, as well as the tissue- and temporal-specific pattern of expression of the transgenes in transformed plants can vary depending on the position of their insertion into the nuclear genome. Such positional effects are well known in the art. For this reason, several nuclear transfoπnants should be regenerated and tested for expression of the transgene.

Methods:

The nucleic acids and polypeptides of the present invention can be used in any one of a number of methods whereby the protein products can be expressed in coffee plants in order that the proteins may play a role in the enhancement of the flavor and/or aroma of the coffee beverage or coffee products ultimately produced from the bean of the coffee plant expressing the protein.

There is a strong correlation between the sucrose concentration in green beans and high quality coffee (Russwurm, 1969; Holscher and Steinhart, 1995 ; Badoud, 2000; Illy and Viani, 1995; Leloup et al., 2003). Improvement of coffee grain sucrose content 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 sucrose metabolism-related genes in coffee, in accordance with the

present invention. For example, the sucrose metabolism enzyme-encoding genes may be genetically mapped and Quantitative Trait Loci (QTL) involved in coffee flavor can be identified. It would be then be possible to determine if such QTL correlate with the position of sucrose related genes. It is also possible to identify alleles (haplotypes), for genes affecting sucrose metabolism and examine if the presence of specific haplotypes are strongly correlated with high sucrose. These "high sucrose" markers can be used to advantage in marker assisted breeding programs. A third advantage of isolating polynucleotides involved in sucrose metabolism is described in detail in the Examples. It is to generate expression data for the genes during coffee bean maturation in varieties with high and low sucrose levels. This information is used to direct the choice of genes to use in genetic manipulation aimed at generating novel transgenic coffee plants that have increased sucrose levels in the mature bean, as described in detail below.

In one aspect, the present invention features methods to alter the sucrose metabolizing enzyme profile, or sugar profile, in a plant, preferably coffee, comprising increasing or decreasing an amount or activity of one or more sucrose metabolizing enzymes in the plant. For instance, in one embodiment of the invention, a sucrose metabolizing enzyme-encoding gene under control of its own expression-controlling sequences is used to transform a plant for the purpose of increasing production of that sucrose metabolizing enzyme in the plant. Alternatively, a sucrose metabolizing enzyme-encoding region is operably linked to heterologous expression controlling regions, such as constitutive or inducible promoters.

In view of the fact that it has been possible to increase the sucrose levels in the pericarp of tomato by the constitutive over-expression of SPS, one preferred embodiment of the present invention comprises transforming coffee plants with an SPS -encoding polynucleotide, such as SEQ ID NO:2, for the purpose of over-producing that coffee SPS in various tissues of coffee. In one embodiment, coffee plants are engineered for a general increase in SPS activity, e.g., through the use of a promoter such as the RuBisCo small subunit (SSU) promoter or the CaMV35S promoter. In another embodiment designed to limit the effects of over-expressing SPS only to the sink organ of interest, i.e., the grain, a grain- specific promoter may be utilized, particularly one of the Coffea grain-specific promoters described above.

The sucrose profile of a plant may be enhanced by modulating the production, or activity, of one or more sucrose metabolizing enzymes in the plant, such as coffee.

Additionally, plants expressing enhance sucrose levels may be screened for naturally- occurring variants of the sucrose metabolizing enzymes. For instance, loss-of-function (null) mutant plants may be created or selected from populations of plant mutants currently available. It will also be appreciated by those of skill in the art that mutant plant populations may also be screened for mutants that over-express a particular sucrose metabolizing enzyme, utilizing one or more of the methods described herein. Mutant populations can be made by chemical mutagenesis, radiation mutagenesis, and transposon or T-DNA insertions, or targeting induced local lesions in genomes (TILLING, see, e.g., Henikoff et al., 2004, Plant Physiol. 135m: 630-636; Gilchrist & Haughn, 2005, Curr. Opin. Plant Biol. 8(2): 211-215). The methods to make mutant populations are well known in the art.

Of particular interest are mutants of sucrose metabolizing enzymes that have select mutations that alter the post-translational modification of the enzyme, which may affect the enzymatic activity or substrate specificity of the enzyme. Post-translational modification is understood in the art to include a number of modifications to a protein that occurs in eukaryotic cells after translation of the protein and can include, among others, glycosylation, alkylation, and phosphorylation of the protein. In some examples, the sucrose metabolizing enzyme SPS, which has been found to have potential phosphorylation sites at Serl50, Ser221 and Ser415, may have phosphorylation sites removed by the introduction of point-mutations at any one or a combination of the potential phosphorylation sites. Through these point- - mutations, the phosphorylation pattern of the enzyme can be modified, thus modifying the activity of the enzyme. Serl50 is thought to be a regulation site for the enzyme SPS; thus, by removing this phosphorylation site by site-directed mutagenesis, the activity of SPS may be enhanced. Additionally, Ser415 is thought to have an antagonizing relationship to the regulatory effect of phosphorylation of Serl50; therefore, removal of this phosphorylation site (Ser415) could further regulate the activity of SPS. Further, phosphorylation at Ser221 of SPS is thought to also inhibit the activity of SPS. Removal of this phosphorylation site (Ser221) could also enhance the activity of SPS.

The nucleic acids of the invention can be used to identify mutant forms of sucrose metabolizing enzymes in various plant species. In species such as maize or Arabidopsis, where transposon insertion lines are available, oligonucleotide primers can be designed to screen lines for insertions in the sucrose metabolizing enzyme genes. Through breeding, a

plant line may then be developed that is heterozygous or homozygous for the interrupted gene.

A plant also may be engineered to display a phenotype similar to that seen in null mutants created by mutagenic techniques. A transgenic null mutant can be created by a expressing a mutant form of a selected sucrose metabolizing enzyme protein to create a "dominant negative effect." While not limiting the invention to any one mechanism, this mutant protein will compete with wild-type protein for interacting proteins or other cellular factors. Examples of this type of "dominant negative" effect are well known for both insect and vertebrate systems (Radke et al, 1997, Genetics 145: 163-171; Kolch et al., 1991, Nature 349: 426-428).

Another kind of transgenic null mutant can be created by inhibiting the translation of sucrose metabolizing enzyme-encoding mRNA by "post-transcriptional gene silencing." The sucrose metabolizing enzyme-encoding gene from the species targeted for down-regulation, or a fragment thereof, may be utilized to control the production of the encoded protein. Full- length antisense molecules can be used for this purpose. Alternatively, antisense oligonucleotides targeted to specific regions of the mRNA that are critical for translation may be utilized. The use of antisense molecules to decrease expression levels of a pre-determined gene is known in the art. Antisense molecules may be provided in situ by transforming plant cells with a DNA construct which, upon transcription, produces the antisense RNA sequences. Such constructs can be designed to produce full-length or partial antisense sequences. This gene silencing effect can be enhanced by transgenically over-producing both sense and antisense RNA of the gene coding sequence so that a high amount of dsRNA is produced (for example see Waterhouse et al., 1998, PNAS 95: 13959-13964). In this regard, dsRNA containing sequences that correspond to part or all of at least one intron have been found particularly effective. In one embodiment, part or all of the sucrose metabolizing enzyme-encoding sequence antisense strand is expressed by a transgene. In another embodiment, hybridizing sense and antisense strands of part or all of the sucrose metabolizing enzyme-encoding sequence are transgenically expressed.

In another embodiment, sucrose metabolizing enzyme-encoding genes may be silenced through the use of a variety of other post-transcriptional gene silencing (RNA silencing) techniques that are currently available for plant systems. RNA silencing involves the processing of double-stranded RNA (dsRNA) into small 21-28 nucleotide fragments by

an RNase H-based enzyme ("Dicer" or "Dicer-like"). The cleavage products, which are siRNA (small interfering RNA) or miRNA (micro-RNA) are incorporated into protein effector complexes that regulate gene expression in a sequence-specific manner (for reviews of RNA silencing in plants, see Horiguchi, 2004, Differentiation 72: 65-73; Baulcombe, 2004, Nature 431: 356-363; Herr, 2004, Biochem. Soc. Trans. 32: 946-951).

Small interfering RNAs may be chemically synthesized or transcribed and amplified in vitro, and then delivered to the cells. Delivery may be through microinjection (Tuschl T et ah, 2002), chemical transfection (Agrawal N et ah, 2003), electroporation or cationic liposome-mediated transfection (Brummelkamp TR et ah, 2002; Elbashir SM et ah, 2002), or any other means available in the art, which will be appreciated by the skilled artisan. Alternatively, the siRNA may be expressed intracellularly by inserting DNA templates for siRNA into the cells of interest, for example, by means of a plasmid, (Tuschl T et ah, 2002), and may be specifically targeted to select cells. Small interfering RNAs have been successfully introduced into plants. (Klahre U et a , 2002).

A preferred method of RNA silencing in the present invention is the use of short hairpin RNAs (shRNA). A vector containing a DNA sequence encoding for a particular desired siRNA sequence is delivered into a target cell by anycommon means. Once in the cell, the DNA sequence is continuously transcribed into RNA molecules that loop back on themselves and form hairpin structures through intramolecular base pairing. These hairpin structures, once processed by the cell, are equivalent to siRNA molecules and are used by the cell to mediate RNA silencing of the desired protein. Various constructs of particular utility for RNA silencing in plants are described by Horiguchi, 2004, supra. Typically, such a construct comprises a promoter, a sequence of the target gene to be silenced in the "sense" orientation, a spacer, the antisense of the target gene sequence, and a terminator.

Yet another type of synthetic null mutant can also be created by the technique of "co- suppression" (Vaucheret et al., 1998, Plant J. 16(6): 651-659). Plant cells are transformed with a copy of the endogenous gene targeted for repression. In many cases, this results in the complete repression of the native gene as well as the transgene. In one embodiment, a sucrose metabolizing enzyme-encoding gene from the plant species of interest is isolated and used to transform cells of that same species.

Mutant or transgenic plants produced by any of the foregoing methods are also featured in accordance with the present invention. Preferably, the plants are fertile, thereby

being useful for breeding purposes. Thus, mutant or plants that exhibit one or more of the aforementioned desirable phenotypes can be used for plant breeding, or directly in agricultural or horticultural applications. They will also be of utility as research tools for the further elucidation of the participation of sucrose metabolizing enzymes and its affects on sucrose levels, thereby affecting the flavor, aroma and other features of coffee seeds. Plants containing one transgene or a specified mutation may also be crossed with plants containing a complementary transgene or genotype in order to produce plants with enhanced or combined phenotypes.

The 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

Plant Material. Tissues from either leaves, flowers, stem, roots, or cherries were harvested at different stages of development from Coffea arabicaL. cv. Caturra T-2308 grown under greenhouse conditions (25°C, 70% RH) in Tours, France, and from Coffea canephora (robusta) BP-409 grown in the field at the Indonesian Coffee and Cacao Research Center (ICCRI), Indonesia. FRT05 (Robusta) and CCCAl 2 (Arabica) were obtained from trees cultivated in Centre Quito, Ecuador. The fruit was harvested at defined stages and frozen immediately in liquid nitrogen, and then packaged in dry ice for transport. Tissues were stored at -80 ⁰ C until use.

Genomic DNA preparation. Leaves from BP-409 were harvested from greenhouse- grown trees at Tours, France. Tissue was frozen immediately in liquid nitrogen and reduced in fine powder. Genomic DNA was prepared according to Crouzillat et al., 1996.

PCR amplification of partial coffee SPS Gene. Degenerated oligonucleotides SPS- 3 (5'ggNcgNgaYtctgaYacNggtgg3') (SEQ ID NO.:20) and SPS-4 (5'tggacgacaYtcNccaaaNgcYttNac3') (SEQ ID NO.:21) were made from the conserved sequence of sucrose-phosphate synthase deduced from the alignment set forth in Figure 4 and used as primers in PCR amplification. PCR reactions were performed in a 50 μl reaction volume with 100 ng genomic DNA, 0.5 μM of each primer, 200 μM of dNTPs, IX Taq polymerase buffer and 1 U of TaqDNA polymerase (TAKARA). After a pre-denaturing step

at 94°C for 5 min, the amplification consisted of 30 cycles of 1 min at 94 ⁰ C, 1 min at 12 different temperatures (from 45°C to 56 ⁰ C) and 2 min at 72°C. The resulting PCR fragments were separated and purified by agarose gel electrophoresis. PCR fragment from the major bands was purified, cloned and sequenced.

Isolation of CcSPSl and CcSPS2 partial cDNA sequences. In order to verify if CcSPSl and CcSPS2 genes were expressed, specific primers were designed based on potential coding sequences identified on the partial genomic CcSPSl and CcSPS2 sequences (SEQ ID NOS: 4 and 5, respectively). Two sets of primers, cDNACl-1 ( ⁵ ^ACTTGCAAGGGCTTTAGGT ^3' ) (SEQ ID NO.:22), cDNACl-2 ( ⁵ ^AGGGCTAGTATCATAGGCT ^3' ) (SEQ ID NO.:23)and cDNADl-1 ( ⁵ ^GCTTGCTAAGGCACTTGCT ^3' ) (SEQ ID NO.:24), cDNADl-2

( ^5' CAATGCTAGAATCATTGGCT ^3' ) (SEQ ID NO.:25) were used to amplify partial CcSPSl and CcSPS2 cDNA sequences respectively by PCR using various cDNA samples prepared as described below.

Universal Genome Walker. Genomic DNA from BP409 was hydrolyzed with four different restriction enzymes (Dral, EcoRV, Pvul, Stul) and the resulting fragments were ligated blunt-end to the Genome Walker Adaptor provided by the Universal Genome Walker kit (BD Biosciences). Both reactions were carried out in accordance with the kit user manual. The four libraries were then employed as templates in PCR reactions using SPS-GSP (gene- specific primers) (Table 1) The reaction mixtures contained 1 μl of GenomeWalker library template, 10 nmol of each dNTP, 50 pmol of each primer and 2.5 units of DNA polymerase in a final volume of 50 μl with the appropriate buffer. The following conditions were used for the first PCR: after pre-denaturing at 95 ⁰ C for 2 min, the first seven cycles were performed at a denaturing temperature of 95 ⁰ C for 30 s, followed by an annealing and elongation step at 72°C for 3 min. A further 35 cycles were carried out, changing the annealing/elongation temperature to 67 ⁰ C for 3 min. Products from the first amplification using the primer pair APl/Cl-GW (Genome Walker) served as template for the second PCR using AP2/C1-GWN (Genome Walker Nested primer), with AP2 and Cl-GWN as primers. The second PCR used 2 μl of the first amplification reaction (undiluted and different dilutions up to 1:50), and was performed as described above for the first reaction, with the exception that the second reaction used only 25 cycles of amplification. The resulting PCR fragments

were separated and purified by agarose gel electrophoresis. PCR fragment from the major bands was purified, cloned and sequenced.

Table 1. List of rimers used for Genome Walker experiments

DNA sequence analysis. For DNA sequencing, recombinant plasmid DNA was prepared and sequenced according to standard methods. Computer analysis was performed using DNA Star (Lasergene) software. Sequence homologies were verified against GenBank databases using BLAST programs (Altschul et al. 1990). cDNA preparation. RNA was extracted from different tissues i.e. root, stem, leaves, flowers, pericarp and grain at four different maturation stages SG (small green), LG (large green), Y (yellow), R (red), as described previously (Benamor and Mc Carthy, 2003). cDNA was prepared from total RNA and oligo dT(18) (Sigma) as follows: 1 μg total RNA sample plus 50 ng oligo dT was made up to 12 μl final volume with DEPC-treated water. This mixture was subsequently incubated at 7O ⁰ C for 10 min and then rapidly cooled on ice. Next, 4 μl of first strand buffer (5x, Invitrogen), 2 μl of DTT (0.1 M, Invitrogen) and 1 μl of dNTP mix (10 mM each, Invitrogen) were added. These reaction mixes were preincubated at 42 ⁰ C for 2 min before adding 1 μl- Superscript III Rnase H-Reverse transcriptase (200U/μl, Invitrogen). Subsequently, the tubes were incubated at 42 ⁰ C for 50 min, followed by enzyme inactivation by heating at 70 ⁰ C for 10 min. The cDNA samples generated were then diluted one hundred fold and 5 μl of the diluted cDNA were used for Q-PCR.

Full length SPS cDNA amplification. In order to amplify full length CcSPSl cDNA, two primers: cDNACl-am3 (^ATGGCGGGAAATGACTGGATAAAC AGTTAC ^3' ) (SEQ ID NO.:36) and cDNACl-am4 ( ^5' CTAGCTTTTGAGAACCCCTAGCTTTTCCAAC ^3' ) (SEQ ID NO.:37) have been designed based on the CcSPSl genomic sequence. These two primers have been used to perform PCR reaction using methods as described above. The single fragment obtained has been purified from agarose gel, cloned and sequenced.

Quantitative-PCR. TaqMan-PCR was carried out as recommended by the manufacturer (Applied Biosystems, Perkin-Elmer). All reactions contained Ix TaqMan buffer (Perkin-Elmer) and 5mM MgC12, 200μM each of dATP, dCTP, dGTP and dTTP, and 0.625 units of AmpliTaq Gold polymerase. PCR was carried out using 80OnM of each gene specific primers, forward and reverse, and 20OnM TaqMan probe. Primers and probes were designed using PRIMER EXPRESS software (Applied Biosystems, Table 2). Reaction mixtures were incubated for 2 min at 50 ⁰ C, 10 min at 95°C, followed by 40 amplification cycles of 15 sec at 95°C/1 min at 60 ⁰ C. Samples were quantified in the GeneAmp 7500 Sequence Detection System (Applied Biosystems). Transcript levels were determined using rpl39 as a basis of comparison.

Table 2. List of primers and probes used for Q-PCR

Soluble Sugars quantification. Grain tissues were separated from pericarp and hulls. The grain were homogenized in cryogenic grinder with liquid nitrogen and the powder obtained was lyophilized for 48 hours (Lyolab bll, Secfroid). Each sample was weighed and suspended in 70 ml of double-distilled water previously pre-heated to 70 °C, then shaken vigorously and incubated for 30 min at 70 ⁰ C. After cooling to room temperature, the sample was brought to 100 ml by adding doubled-distilled water, and then paper filtered (Schleicher and Schuell filter paper 597.5). Sugars of extracted coffee grain tissues were separated by HPAE-PED according to Locher et al., 1998 using a Dionex PA 100 (4x250 mm) column. Sugar concentration was expressed in g per 100 g of DW (dry weight).

Enzymatic Activity analysis. Sucrose synthase activity was measured according to Lafta and Lorenzen (1995). Sucrose phosphate synthase activity was measured according to Trevanion et al. (2004).

Example 2

Identification of cDNA Encoding Enzymes of Sucrose Metabolism

More than 47,000 EST sequences were identified from several coffee libraries made with RNA isolated from young leaves and from the grain and pericarp tissues of cherries harvested at different stages of development. Overlapping ESTs were subsequently "clustered" into "unigenes" (ie contigs) and the unigene sequences were annotated by doing a BLAST search of each individual sequence against the NCBI non-redundant protein database.

Enzymes directly involved in the synthesis and degradation of sucrose have been widely studied in plants, and especially during fruit, tuber, and seed development in plants such as tomato (Lycopersicon esculentum), potato (Solanum tuberosum) and corn (Zea mays). DNA sequences coding for all known key proteins involved in sucrose synthesis and degradation have been identified and characterized in several species and are available in GenBank. Accordingly, the known sequences of plant enzymes, especially sequences from organisms closely related to coffee (e.g., tomato and potato), were used to find similar sequences present in the above-described EST libraries and in other coffee cDNA libraries. To search the aforementioned EST collection, protein sequences of tomato and potato were used in a fBLASTn search of the "unigene" set 3 as described in Example 1. Those in-sϊlico "unigenes" whose open reading frames showed the highest degree of identity with the

"query" sequence were selected for further study. In some cases, the selected "unigenes" contained at least one EST sequence that potentially represented a full length cDNA clone, and that clone was then selected for re-sequencing to confirm both its identity and the "unigene" sequence.

A. Sucrose synthase CcSS2 (SEQ ID NO:1)

The clone A5-1540, which is highly related to sucrose synthase 2 (SS2) from tomato (Lycopersicon esculentum, NCBI Protein Identifier No. CAA09681), was found in a coffee cDNA collection (as opposed to the EST collection). The protein encoded by A5-1540 clone is 88.6 % identical to SS2 from tomato and is apparently full length (Figure 2). The cDNA insert is 3048 bp long, and is characterized by a 2427 bp ORP which starts at position 248 and finishes position 2668. This sequence is referred as SEQ ID NO 1. The deduced protein (SEQ ID NO: 8) is 805 aa long, with a predicted molecular weight of 92.6 kDa. The protein sequence encoded by the clone A5-1540 was analyzed for similarity to all publicly available protein sequences contained in the NCBI nonredundant database. The resulting alignment of the most closely related sequences is presented in Figure 2. As can be seen from the figure, residues 7-554 of SEQ ID NO: 8 comprises a domain that characterizes members of the sucrose synthase family, and residues 565-727 is a domain that characterizes glycosyl transferase group 1.

As well as being closely related to SS2 of tomato, it is also closely related to potato SS2 (89% identity; NCBI Protein Identifier No.AA034668). Subsequently, unigene # 97089 was found in the EST database, and that sequence was determined to correspond to the same sequence as A5-1540. However, the longest clone in this unigene is over 1,400 nucleotides shorter than the clone A5-1540, and thus the EST database does not appear to contain a full length clone. In monocotyledons (maize and sorghum), SS is encoded by three differentially expressed nonallelic loci, susl, sus2 and sus3 (Chourey et al., 1991, Huang et al., 1996, Carlson et al. 2002). Most dicotyledonous species contain two nonallelic SS genes, which are functional analogs of two classes of SS genes from monocotyledons (Fu et al., 1995). Homology results show that the coffee sequence is closest to the SS2 sequence of potato and the protein encoded by clone A5-1540 therefore was designated CcSS2, for Coffea canephora sucrose synthase 2, herein.

B. Sucrose phosphate synthase CcSPSl (SEQ ID NO:2)

The protein sequence of sucrose phosphate synthase (SPSLEl, NCBI Protein Identifier No. AAC24872) from tomato {Lycopersicon esculentum) was used to perform a similarity search of the EST-based unigene set using the tBLASTn algorithm. No unigene was found that could potentially code for an SPS protein.

Due to lack of a match in the available database, a partial sequence from the Coffea canephora BP-409 genome was amplified using degenerate oligonucleotides. By alignment of different SPS protein sequences, it was possible to identify a highly conserved domain and to design degenerate primers corresponding to the protein sequence encoded at the end of exon 4 and at the beginning of exon 7 (Fragment Cl in Figure 3).

Two different PCR fragments of 1500 and 2000 bp, respectively, were amplified using PCR and degenerate oligonucleotides. After sequencing of both genomic sequences, an alignment of putative encoded protein sequences with the tomato protein sequence SPSLEl showed isolation of partial sequences from two different coffee SPS genes, CcSPSl and CcSPS2. The fragments corresponding to CcSPSl and CcSPS2 partial sequence were 1937 and 1564 bp long, respectively . The protein sequences encoded by partial clones of CcSPSl and CcSPS2 were found to share a high degree of homology. Introns 4, 5 and 6 were shorter for CcSPS2, thus explaining the difference in size between the two amplified fragments (data not shown). Preliminary expression analysis indicated that CcSPS2 was not expressed, while CcSPSl was expressed in various tissues, including grain. Therefore, the CcSPSl gene was examined further.

Using several rounds of primer directed genome walking using the Genome Walker™ technique, a full length genomic sequence for the CcSPSl gene was amplified. A schematic representation of the CcSPSl gene is shown in Figure 3. The gene is characterized by 13 exons and 12 introns. The CcSPSl gene is 7581 bp long (from initiation codon ATG to stop codon TAG) is referred to as SEQ ID NO:6. Using specific primers deduced from CcSPSl genomic sequence, the CcSPSl full length cDNA was amplified by RT-PCR. Several RNA samples were used, positive amplification corresponding to the full length cDNA sequence was only obtained using RNA extracted from pericarp at yellow stage from robusta. The CcSPSl cDNA is 3150 bp long and this DNA sequence is referred as SEQ ID NO: 2. The deduced protein, SEQ ID NO: 9, is 1049 aa long, with a predicted molecular weight of 117.9 kDa. The protein sequence encoded by the CcSPSl cDNA shows a very high level of

homology (82.6%) with the tomato SPSLEl protein sequence (Figure 4). In addition, residues 168-439 of SEQ ID NO:9 characterize members of the sucrose phosphate synthase family, and residues 467-644 characterize glycosyl transferases group 1 family members.

C. Sucrose phosphate phosphatase CcSPl (SEQ ID NO:3)

The protein sequence of sucrose phosphatase (SP, NCBI Protein Identifier No. AA033160) from tomato (Lycopersicon esculentum) was used to perform a similarity search of the EST-based unigene set using the tBLASTn algorithm. The ORF of unigene #102159 showed a high degree of homology to the tomato SP sequence and the single EST (cDNA) in this unigene, clone cccll9nl5, was isolated and its insert was fully sequenced. The cDNA insert of cccll9nl5 is apparently full length and was found to be 1721 bp long. This sequence is referred as SEQ ID NO 3. The complete ORF sequence of this clone was 1248 bp long, starting at position 135 and finishing at position 1409. The deduced protein (SEQ ID NO.'IO) was 415 aa long with a predicted molecular weight of 46.7 kDa. Residues 1-408 of SEQ ID NO: 10 characterize members of the sucrose-6F-phosphate phosphohydrolase family. The ORF of cccll9nl5 is 81 % identical to the tomato SP protein. The protein encoded by cccll9nl5 was also analyzed for similarity to all publicly available protein sequences contained in the NCBI nonredundant database. The alignment of sequences showing the highest homologies is presented in Figure 5. Only one distinct unigene has been found in the coffee cDNA libraries. Several species (including maize, tomato, wheat and barley) are known to contain at least two SP genes; Arabidopsis has four and rice three (Lunn et MacRae, 2003). Based on homology results presented here, the cDNA clone cccll9nl5 clone has been renamed CcSPl for C. canephora sucrose phosphatase 1.

Example 3

Control of SPS Activity by Reversible Protein Phosphorylation

A major regulatory site of spinach SPS has been identified as Ser 158 (McMichael et al., 1993). Phosphorylation of Serl58 is both necessary and sufficient for the inactivation of SPS in vitro. Similar results were shown for the phosphorylation of Ser 162 of maize SPS in studies of maize leaves, as well as transgenic tobacco expressing the maize SPS gene (Huber et al., 1995). Although the regulatory phosphorylation sequence of spinach SPS is not

conserved exactly, all sequences available to date were determined to contain a homologous seryl residue.

The CcSPSl deduced protein sequence was aligned with other SPS protein sequences from different species. The serine residue of CcSPSl most likely to be homologous to Ser 158 in spinach was identified as Ser 150 (Figure 4). Most of the residues surrounding the putative phosphorylation site are consistently conserved among the five aligned proteins, e.g., there are basic residues at P-3 (R), P-6 (R) and P-8 (R or K) (numbering relative to Ser, which is position 0). Several, and possibly all, of these conserved residues may be important for recognition by a protein kinase (Huber and Huber, 1996; McMichael et al., 1993). The enzymatic activity of CcSPSl could be modulated by phosphorylation/dephosphorylation of Serl50. In one example, the Serl50 could be replaced with another conservative replacement amino acid by site-directed mutagenesis; thereby, eliminating this phosphorylation site and enhancing SPS activity by eliminating regulation via phosphorylation.

Recent evidence suggests that there may be a second regulatory phosphorylation site at Ser 424 of spinach SPS, which is phosphorylated when leaf tissue is subjected to osmotic stress (Toroser and Huber, 1997). Phosphorylation of Ser 424 in spinach activates the enzyme, perhaps by antagonizing the inhibitory effect of Serl58 phosphorylation. This site was also determined to be widely conserved among species. The homologous site in CcSPSl protein was determined to be Ser 415. The sucrose synthesis activity of SPS could be enhanced by placing coffee in a simulated high osmotic stress environment to facilitate the phosphorylation of Ser415.

A third potential phosphorylation site may also exist, inasmuch as recent results have demonstrated that 14-3-3 proteins can associate with spinach leaf SPS in the presence of Mg ²⁺ . The effect of this specific 14-3-3 protein/SPS interaction was to partially inhibit the SPS activity. It has been proposed that the 14-3-3 protein may function as a scaffold protein to facilitate the interaction of SPS with other proteins. The suggested site of interaction in spinach SPS is Ser 229. The homologous region in CcSPSl is Ser 221 and, notably, this region of the protein is strongly conserved (Figure 4). In one example, the Ser221 could be replaced with another conservative replacement amino acid by site-directed mutagenesis; thereby, eliminating this phosphorylation site and enhancing SPS activity by eliminating regulation via phosphorylation.

In summary, SPS enzymatic activity can be regulated by reversible protein phosphorylation, and three sites have been shown to be involved in enzyme activity regulation of spinach or maize enzyme. By alignment of CcSPSl with SPS from other species, these putative seryl residues have been localized in CcSPSl to Ser 150, Ser 221 and Ser 415.

Example 4

Sugar Accumulation and Enzymatic Activity During Coffee Seed Development

Sugar Quantification. Sugar levels during coffee grain maturation were examined in C. canephora variety FRT05 (robusta) and C. Arabica variety CCCAl 2 (arabica). These two genotypes were chosen because they have been found to possess significantly different levels of sucrose. The amounts of sucrose, glucose and fructose in the FRT05 and CCCAl 2 coffee grain during maturation were measured in samples harvested in parallel. The same samples were also used for the assays of SPS and SuSy activity described below. The results are shown in Figure 6.

At the earliest stage of maturity (stage SG), the main free sugar was found to be glucose, but the concentration is 10 times higher in arabica (14 %) than robusta (1.5 %). At the same stage, fructose concentration was also higher in arabica (1.5 %) than robusta (0.3 %), but at a much lower level than glucose. By the end of grain development, concentrations of glucose and fructose were found to have decreased to very low levels for both species, with only trace levels being detected at the mature red stage (R). The decrease in fructose and glucose was accompanied by an increase in sucrose, which approached 100 % of total free sugars in mature grains, with higher levels found in arabica (9.82 %) than robusta (6.71 %). These results represent only free sugar accumulation and do not include their modified form, e.g., UDP-G, F6-P and S6-P, which are also known to play a role in sucrose metabolism (Figure 1).

SuSy and SPS Enzyme Activity. In parallel to the sugar quantification, sucrose synthase (SuSy) and sucrose phosphate synthase (SPS) enzyme activities were studied in order to determine if there might be a strong correlation between free sugar accumulation and these particular enzyme activities and to elucidate the reason CCCAl 2 (Arabica) accumulates 30 % more sucrose than FRT05 (robusta).

The enzymatic activities of SuSy and SPS were determined similarly for each of the same development stages. SuSy (EC 2.4.1.13) catalyzes the reversible cleavage of sucrose in the presence of UDP to form UDP-glucose and fructose, while SPS (EC 2.3.1.14) catalyzes the synthesis of sucrose phosphate and UDP starting from fructose 6-phosphate and UDP- Glucose (Figure 1).

Low SuSy activity was observed in early stage of development (stage SG), with the activity being almost two times higher in arabica (0.007 U) than robusta (0.004) (Figure 6). SuSy activity rose drastically between SG and LG stage and reached a peak of 0.069 U for arabica and 0.12 U for robusta. Again, SuSy activity was twice as high for robusta as compared with arabica at the LG stage. In the later stage of development, the SuSy activity declined dramatically for both species to reach approximately similar low levels of activity at the Y stage. Between Y and R stages, SuSy activity remained constant but weak for arabica as well as for robusta.

Overall, the SuSy activity was clearly higher at all stages in robusta than in arabica. The profiles of SuSy activity during both arabica and robusta grain development were similar to those seen in various other plants, such as tomato and maize. For those species, it has been shown that SuSy activity is highly correlated with sucrose unloading capacity from the phloem (phenomenon also called sink strength; Sun, et al., 1992; Zrenner et al., 1995). If this correlation exists in coffee grain, this implies that the sink strength of robusta should be higher at the large green stage of robusta versus the same stage of arabica. Interestingly, although the peak of SuSy activity reached its highest point between SG and LG stage in both species, the sucrose concentrations of both were not drastically increased. This suggests either that sucrose is not re-synthesised immediately after import, or that sucrose is being rapidly funnelled into another pathway.

The activity pattern observed for SPS activity during coffee seed maturation was found to be completely different for arabica and robusta (Figure 6). At the earliest stage (SG), SPS activity in robusta (0.02 U) was 2.5-fold higher than that observed for arabica (0.008 U). In robusta, SPS activity was seen to decrease to undetectable levels at the Y stage then rise again to the levels seen in the SG stage at R stage. In contrast, for arabica, SPS activity rose sharply between SG and LG stages, reaching an activity of 0.04 U, and then continued to rise slowly during the final maturation process, reaching 0.052 U at stage R. The fluctuation of SPS activity appeared to be quite high for robusta, while, in contrast, the

SPS activity rose gradually during grain maturation to reach a relatively high level at the R stage in arabica. The difference in the SPS activity levels during grain maturation is likely to be an important contributing factor that leads to sucrose accumulation that is 30 % higher in arabica than robusta.

Example 5

CcSS2, CcSPSl and CcSPl mRNA Expression at Different Stages of Coffee Grain

Maturation

To determine if any correlation existed between the enzymatic activity fluctuations seen for SuSy and SPS and the expression of these genes during coffee bean maturation, the expression of the three genes CcSS2, CcSPSl and CcSPl during T2308 (C. arabica, arabica) and BP-409 (C canephora, robusta) grain development was characterized. For comparative purposes, the expression of these genes in different coffee tissues, such as leaf, flower and root, was also examined. It is noted that gene expression analysis was not carried out on the same genotypes as those used for enzymatic activities, but comparisons were still made between arabica and robusta.

RNA was extracted from BP-409 and T2308 coffee cherries at four different maturation stages characterized by size and color, i.e. SG (small green), LG (large green), Y (yellow) and R (red or mature). For each stage, pericarp and grain were separated before total RNA was extracted as described in Example 1. Total RNA was also extracted from other tissues (leaf, root and flower). Gene expression was analyzed by performing real time RT- PCR (TaqMan, Applied Biosystems). Relative transcript levels were quantified against an endogenous constitutive transcript rpl39. The gene specific primers and the TaqMan probes used are listed in Example 1.

The CcS S2 transcript was highly expressed in robusta grain at the earliest stage of development (6 U of RQ). The level of CcSS2 mRNA then decreased gradually during coffee grain maturation until being equivalent to a value of 0.3 U of RQ at the mature stage (R). A relatively similar expression pattern was seen for arabica grain, although the absolute levels were different. The relative amount of expression for CcSS2 was higher in robusta than arabica grain at the SG stage, equivalent at LG stage for both species, and lower in robusta than arabica at the Y stage. Transcript accumulation was slightly greater in arabica than robusta at the mature stage (R). Except in the small green stage, low levels of CcSS2

accumulate in the pericarp of robusta and arabica cherries. Similar results (Wang et al, 1994; Carlson et al., 2002) were obtained previously in tomato and maize, in both cases there was an early accumulation of SuSy mRNA, followed by decreasing levels of transcripts being detected as fruit and grain maturation progressed. Significant levels of CcSS2 transcripts were also detected in other coffee tissues such as root, flower and leaf for both species.

The CcSPSl transcript is expressed at very low levels compared to CcSS2 mRNA, with the highest RQ observed being 0.07 U in arabica (Figure 6). CcSPSl transcripts were almost undetectable during all stages of robusta grain maturation examined, and the highest expression detected in robusta was in yellow pericarp and flower (0.02 U). The level of CcSPSl transcripts in the robusta root and leaf tissues were below detection. Interestingly, CcSPSl transcripts were detected in all the tissues examined for arabica, especially in the flower, grain and pericarp. In arabica, the transcript level increased 10-fold between SG (0.005) and Y stage (0.05 U). In mature grain stage, the level was slightly lower (0.04 U). Overall, the results obtained indicate that CcSPSl mRNA expression is significantly higher in arabica than robusta. This main result correlates well with the differences in SPS activity, presented earlier, with the detectable SPS activity being significantly higher in arabica than in robusta.

CcSPl transcript accumulation was very low in grain and pericarp at all the maturation stages examined for both species, although expression was slightly higher in arabica than robusta (Figure 6).

Generally, arabica grains accumulate more sucrose than do robusta grains. To summarize the results set forth above, chemical analysis showed that CCCA 12 (Arabica) accumulated 30 % more sucrose in the mature grain than FRT05 (robusta). The activity of SuSy and SPS was also determined for both species during coffee bean maturation. Notably, SuSy activity was found to be higher in robusta than arabica. The peak of activity was found at the LG stage for both species. The rapid growth phase of coffee fruit development is between SG and LG stage, a phase that correlates well with the highest level of SuSy activity. Results in other systems have shown that SuSy activity is highly correlated with sucrose unloading capacity from the phloem at the earliest stages of tomato fruit development (Sun, et al., 1992; Zrenner et al., 1995; N'tchobo, 1999; Wang et al., 1993). The postulate that the level of SuSy activity indicates the level of sink strength may suggest that the sink strength is higher in robusta than arabica. SPS activity has also been determined at the same stages of

grain maturation. However, SPS activity did not follow the same schema in robusta and arabica during coffee grain maturation. While SPS activity fluctuated during robusta grain development, the activity rose steadily during early arabica grain development and stayed at relatively high levels until the end of maturation. Without intending to be limited by any explanation of mechanism, these observations suggest a mechanism in which the steady increase in SPS activity seen during arabica grain maturation may account for at least part of the difference in sucrose concentration found in mature arabica versus robusta grain. CcSS2 and CcSPSl mRNA accumulation in the developing grain was also consistent with the activity levels detected for the respective enzymes. For SuSy, the level of CcS S2 transcript accumulation was seen to increase up to the large green stage then fall as the grain maturity continued. The level of CcSPS-Cl transcripts rose consistently, albeit slightly, as arabica grain maturation progressed, while the level of transcripts was much lower in the grain of robusta at all stages examined. Again, the expression data obtained for CcSS2 and CcSPSl supports the notion that SPS activity could be the limiting step for re- synthesis of sucrose during final steps of coffee grain maturation, especially in robusta grain.

In the perspective of genetically improving robusta coffee quality, the SPS enzyme therefore may be a key factor for generating higher final sucrose concentrations in the mature grain. Alterations in carbon partitioning in plants, and most particularly improvement of sucrose levels in sink organs, have been accomplished in other plant species. For instance, tomato plants were transformed with a construct comprising a maize SPS cDNA under the control of the SSU promoter (Rubisco small subunit promoter) (Worrell, et al., 1991; Galtier et al. 1993; Foyer and Ferrario, 1994; Micallef, et al., 1995; Van Assche et al., 1999; Nguyen- Quoc et al., 1999). The total SPS activity in the leaves of the transformed plants was six times greater than that of the controls and the total SPS activity in the mature fruit from the transformed plants was twice than that of untransformed controls. The combination of overexpression, combined with possible de-regulation of enzyme activity in the transgenic tissue were thought to contribute to the overall increase in SPS activity. The increase in SPS activity was also accompanied by a significant increase (25%) in total overall SuSy activity in 20 day old tomato fruit. In this case, SuSy activity was measured with an assay in the direction of sucrose breakdown (Nguyen-Quoc et al.; 1999). Fruit from these transgenic tomato lines also showed higher sugar content (36 % increase) compared to untransformed plants (Van Assche et al., 1999). Biochemical studies have also shown that the high levels of

the corn SPS activity in the plants caused a modification of carbohydrate portioning in the tomato leaves with an increase of sucrose/starch ratios and also a strong improvement in the photosynthetic capacity. The tomato plants appeared to tolerate the elevated levels of SPS as there were no apparent detrimental growing effects. In studies by others, plants transformed with the construct 35SCaMV-SPS (35 S Cauliflower Mosaic Virus) were found to have three to five times more total SPS activity in leaves than in wild-type plants, but tomato fruit obtained from those particular transformants did not show any increase in SPS activity (Laporte et al. 1997; Nguyen-Quoc et al. 1999). Thus, it appears that the choice of promoter can influence the ultimate effect of transformation of plants with a heterologous SPS gene.

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