2. Description of Related Art Volatile esters are major constituents in many floral aromas (Knudsen et al., 1993). In the moth-pollinated flowers of Clarkia breweri, an annual native to the coastal range of California, benzylacetate constitutes up to 40% of the total scent output. Two other esters, benzylbenzoate and methylsalicylate, contribute about 5% each (Raguso and Pichersky, 1995). Since benzenoid esters, and in particular benzylacetate, are emitted by a majority of moth-pollinated flowers, it was inferred that the moth pollinators find such benzenoid esters attractive (Knudsen and Tollsten 1993). Raguso et al. (1996) recently tested this hypothesis by exposing captured individuals of Hyles lineata, the nocturnal hawkmoth that pollinates C. breweri, to various volatiles and measuring the electroantennogram responses. They showed that responses following exposure to benzylacetate and methylsalicylate were among the strongest recorded.

Although some floral volatile esters, such as methylsalicylate and methyljasmonate, have important functions in some vegetative processes as well (Farmer and Ryan, 1990 ; Shulaev et al., 1997), the biochemical synthesis of such esters has received little attention. Thus, despite the importance of floral scent to plant reproduction and evolution and flavor to the desirability of a plant product, the

biochemical and genetic basis of scent production has received little attention.

Previous reports have failed to identify and purify specific enzymes involved in the biosynthesis of scent components in flowers. Since many scent components also are found in floral tissues in bound, non-volatile forms such as glycosides, it was originally hypothesized that scent compounds could possibly be synthesized elsewhere in the plant, bound into glycosides, and then transported to the emitting part of the flower, where they could be broken down to release the volatile components (Ackermann et al., 1989; Watanabe et al., 1993). However, direct and reproducible evidence of the transport of free scent constituents or their glycosides from vegetative tissue to floral tissue is lacking.

Elucidation of biosynthetic pathways of production of fragrance-, scent-and flavor-conferring compounds, as well as the cloning of genes involved in these pathway, would allow for the production of transgenic plants with enhanced profiles of scent components and would be commercially important. Such plants would have improved flavor and fragrance and represent a significant advance to agriculture. To date, however, reaching the goal of producing such plants has been severely limited by the general lack of information regarding the biosynthetic pathway (s) involved and genes which encode enzymes in such pathway (s).

SUMMARY OF THE INVENTION Thus, it is an objective of the present invention to provide DNA compositions and their use in manipulating the biosynthesis of flavoring compounds in plants.

More specifically, the present invention identifies a novel gene and protein involved in the benzenoid pathway. Methods and compositions for the use of BEAT are disclosed herein.

Thus, in order to achieve the objectives outlined herein, there is provided an isolated nucleic acid comprising a nucleic acid segment coding for benzylalcohol acetyl transferase (BEAT) or an active fragment thereof. In specific embodiments, the nucleic acid segment encodes full length BEAT. In more particular embodiments, the

BEAT protein has the sequence of SEQ ID N0: 2. In an alternative embodiments, the nucleic acid segment has the sequence of SEQ ID NO: 1. In still further alternative embodiments, the nucleic acid segment has the sequence of SEQ ID N0: 16, SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19 or any other variant thereof.

In a specific embodiments of the present invention, there is provided an isolated and purified BEAT protein. More particularly the BEAT protein has the sequence of SEQ ID N0: 2. In certain other embodiments, the BEAT may have a protein sequence of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID N0: 12, SEQ ID NO: 13 or any other variant thereof. By"any other variant thereof'the present invention contemplates that the BEAT protein need not have the exact sequences disclosed so long as the protein has BEAT activity as described herein.

In another aspect of the present invention there is provided an expression vector comprising a nucleic acid segment coding for benzylalcohol acetyl transferase (BEAT) and a promoter operatively linked to the nucleic acid segment. In more defined aspects, the promoter may be selected from the group consisting of 35S promoter, E8 promoter and CHS-A promoter. In other embodiments, the promoter may be a fruit-specific promoter or a leaf-specific promoter. In particular embodiments, the expression vector may further comprise an origin of replication and a polyadenylation signal.

Also contemplated by the present invention is a method for increasing the synthesis of benzyl acetate in a plant cell comprising the steps of providing a plant cell; contacting the plant cell with a nucleic acid segment coding for benzylalcohol acetyl transferase (BEAT) and a promoter operatively linked to the nucleic acid segment; and selecting a cell having the nucleic acid stably integrated into is genome.

In specific embodiments, the plant cell may be independently, a monocot or a dicot. More particularly, the dicot plant cell may be a tomato cell, a petunia cell, or a snapdragon cell. In more particular aspects, the contacting may comprise independently, microprojectile bombardment, electroporation or Agrobacterium-

mediated transformation. In other embodiments, the selecting may comprise identifying a cell having drug resistance. More particularly, the drug resistance is kanamycin or hygromycin resistance.

The present invention further provides a transgenic plant cell having, incorporated into its genome, a nucleic acid segment coding for benzylalcohol acetyl transferase (BEAT) and a promoter operatively linked to the nucleic acid segment.

More particularly the plant cell may be a monocot or a dicot.

Further, the present invention also contemplates a transgenic plant having, incorporated into the genome of cells of the plant, a nucleic acid segment coding for benzylalcohol acetyl transferase (BEAT) and a promoter operatively linked to the nucleic acid segment.

The present invention also provides a method for decreasing the benzylalcohol acetate content of a plant cell comprising the steps of providing a plant cell; contacting the plant cell with a nucleic acid segment coding for benzylalcohol acetyl transferase (BEAT) and a promoter operatively linked to the nucleic acid segment; and selecting a cell having the nucleic acid stably integrated into its genome. The contacting may comprise microprojectile bombardment, electroporation or Agrobacterium-mediated transformation.

In yet another embodiment, the present invention provides a recombinant host cell comprising a nucleic acid segment encoding BEAT. The recombinant host cell may be, further defined as a prokaryotic cell, a eukaryotic cell, a plant cell or a bacterial cell. In preferred embodiments, the bacterial cell may be an E. coli, B. subtilis, B. megaterium or a Pseudomonas sp. cell. In particular embodiments, the host cell expresses the nucleic acid segment to produce a BEAT protein or peptide.

More particularly, the BEAT protein or peptide comprises a sequence of SEQ ID NO: 2. In other embodiments, the BEAT protein or peptide comprises an amino acid sequence encoded by a nucleic acid sequence from SEQ ID NO: 1.

The present invention further provides a polyclonal antisera that binds immunologically to a BEAT protein or peptide. In other specific embodiments, the present invention provides a monoclonal antibody that binds immunologically to a BEAT protein or peptide.

Also provided is a method of using a nucleic acid segment that encodes a BEAT protein or peptide, comprising the steps of introducing a recombinant vector into a host cell wherein the vector comprises a BEAT protein or peptide-encoding DNA segment positioned under the control of a promoter; culturing the host cell under conditions effective to allow expression of the encoded BEAT protein or peptide; and collecting the expressed BEAT protein or peptide.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1A, FIG. 1B and FIG. 1C. Emission of benzenoid esters from C. breweri flowers as measured by headspace collection at 12 hour intervals and GC- MS analysis. FIG. 1A. Emission of benzylacetate. Data are means SE (n > 3).

FIG. IB. Emission of benzylbenzoate I. Data are means SE (n : 3). FIG. 1C.

Emission of methylsalicylate. Data are means SE (n > 3).

FIG. 2. Emission of benzenoid esters from C. breweri flowers and flower parts on day 1 of anthesis, as measured by headspace collection at a 24 hour interval and GC-MS analysis. Data are means SE (n = 5-8).

FIG. 3. The reactions catalyzed by BEAT and SAMT.

FIG. 4A and FIG. 4B. Levels of different BEAT and SAMT activities in different parts of the flower during the lifespan of the flowers. FIG. 4A. BEAT activity. For each data point, flowers from three different plants were combined for each assay, and 2-3 enzyme assays were conducted and the mean obtained. Standard errors for data points on day 1 of anthesis are given in Table 2. Standard errors for other time points are similar. The pkat unit is defined in Table 2. FIG. 4B. SAMT activity. For each data point, flowers from three different plants were combined for each assay, and 2-3 enzyme assays were conducted and the mean obtained. Standard errors for data points on day 1 of anthesis are given in Table 2. Standard errors for other time points are similar. The pkat unit is defined in Table 2.

FIG. 5: Purification of BEAT from petal tissue of C. breweri and from E. coli cells expressing plant BEAT.

FIG. 6A, FIG. 6B, FIG. 6C, FIG. 6D and FIG. 6E: GC analysis of BEAT activity in vitro and in vivo. FIG. 6A Benzylacetate standard. FIG. 6B Analysis of product formed in vitro in a reaction catalyzed by plant-purified enzyme. FIG. 6C Analysis of product formed in vitro in a reaction catalyzed by the plant enzyme purified from E. coli cells expressing BEAT cDNA. FIG. 6D Analysis of spent media in which E. coli cells expressing BEAT DNA grew. FIG. 6E Analysis of spent media in which E. coli cells which did not possess BEAT cDNA grew. Toluene was added to

all samples for quantitation purpose. Peaks were analyzed by mass-spectrometer for identification.

FIG. 7: Nucleotide sequence of BEAT cDNA clone. The predicted protein sequence is shown below the nucleotide sequence. Numbers on right refer to the nucleotide sequence, numbers on left refer to the protein sequence. Peptide sequences obtained in this study are underlined. The conserved motif found in other acetyl CoA- binding proteins is shown in black boxes with white letters.

FIG. 8A and FIG. 8B: Expression of BEAT in flower parts. FIG. 8A Northern blot hybridization of RNA from different tissues of high-activity plants.

Each lane contained 7 u. g total RNA. The blot was rehybridized with an 18S rDNA probe to standardize samples. FIG. 8B Same as FIG. 8A but with RNA from low- activity plants.

FIG. 9A and FIG. 9B: Expression of BEAT throughout the lifespan of the flower. FIG. 9A Northern blot hybridization with RNA from petals at different stages of development. Total RNA (3 u. g) was obtained from different stages of flowers with high BEAT activity. FIG. 9) Plot of the variation in levels of petal BEAT mRNA.

Values were obtained by scanning blots with a phosphoimager. Each point is the average of 2 studies (including the one shown in FIG. 9A, and values were corrected by standardizing for 18S RNA levels.

FIG. 10: Segregation of F2 individuals for levels of BEAT activity. All plants were grown under identical conditions, and all enzyme extractions were carried out on petals on the day of anthesis. From each individual, three flowers were assayed separately, and results were averaged. Standard error in most cases was <10%. The asterisk above the bar at the 575 fkat mark indicates the individual with high BEAT activity shown in FIG. 11 to be heterozygous for BEAT alleles.

FIG. 11A and FIG. 11B: DNA blots analyses. FIG. 11A A blot of genomic DNA of the low-activity parent, digested with five restriction enzymes and probed with a BEAT cDNA. FIG. 11B Examples of genomic DNA blots of 10 F2 individuals, digested with HindIII and probed with same BEAT cDNA.

FIG. 12A, FIG. 12B and FIG. 12C: Contiguous amino acid sequence of BEAT and its comparison to BEAT 1, BEAT 2 BEAT 3 and BEAT 4.

FIG. 13A, FIG. 13B and FIG. 13C: Relative amount of BEAT in different tissues of C. breweri and C. concinna. FIG. 13A shows the relative amount of BEAT mRNA. FIG. 13B shows the relative amount of BEAT protein. FIG. 13C shows the relative amount of BEAT activity (pkat/mg protein).

FIG. 14A, FIG. 14B and FIG. 14C: Relative amount of BEAT in petal tissue of the two species during the lifespan of the flower. FIG. 14A shows the relative amount of BEAT mRNA. FIG. 14B shows the relative amount of BEAT protein.

FIG. 14C shows the relative amount of BEAT activity (pkat/mg protein).

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS I. The Present Invention In an effort to provide the means for producing plants with altered flavor and fragrance characteristics, the current inventors have sought to exploit and manipulate the biosynthetic pathway of phenylpropanoids. In one aspect, the invention relates to the cloning of a gene responsible for the production phenylpropanoid components of the C. breweri scent. The inventors further demonstrate that the genome of C. concinna also contains BEAT genes, that these genes are expressed in both floral and vegetative tissues, and that the reason why C. breweri flowers produce and emit benzylacetate but C. concinna flowers do not, are complex and involve more than simple control of gene transcription. These findings provide for methodology permitting production of transgenic plants with altered fragrance and flavoring

characteristics as well as altering the scent of plants to better attract insects for pollination.

The fragrance of Clarkia breweri (Onagraceae), a California annual plant, includes 3 benzenoid esters, benzylacetate, benzylbenzoate and methylsalicylate.

Here, the inventors report that petal tissue is responsible for benzylacetate and methylsalicylate emission, whereas the pistil is the main source of benzylbenzoate.

The activities of two novel enzymes: acetyl CoA: benzylalcohol acetyltransferase (BEAT), which catalyzes the acetyl esterification of benzylalcohol, and SAM: salicylic acid carboxyl methyltransferase (SAMT) which catalyzes the methyl esterification of salicylic acid, also were highest in petal tissue and absent in leaves. In addition, the activity of both enzymes in the various floral organs was developmentally and differentially regulated. SAMT activity in petals peaked in mature buds and declined during next few days after anthesis, and it showed strong positive correlation with the emission of methylsalicylate. The levels of BEAT activity and benzylacetate emission in petals also increased in parallel as the buds matured and the flowers opened, but as emission began to decline on the second day after anthesis, BEAT activity continued to increase and remained high to the end of the lifespan of the flower.

The inventors also have developed enzymatic assays to test for the activities of the biosynthetic enzymes catalyzing the formation of methylsalicylate and benzylacetate-SAM: salicylic acid carboxyl methyltransferase (SAMT) and acetyl CoA: benzylalcohol acetyltransferase (BEAT), respectively. The activity of benzoyl- CoA: benzylalcohol benzoyltransferase (BEBT), the hypothetical enzyme that catalyzes the formation of benzylbenzoate (Gross, 1981; Croteau and Karp, 1991), was not tested-for lack of a labeled substrate with a suitably high specific activity.

The activities of SAMT and BEAT follow complex patterns throughout the lifespan of the flower. Overall, the data show that scent production in C. breweri is a complex process that involves spatial and temporal patterns of regulation that are not necessarily identical for all enzymes involved.

Further, the inventors have purified the enzyme acetyl CoA: benzylalcohol acetyl transferase (BEAT), which catalyzes the formation of benzylacetate, from a C. breweri line that has high BEAT activity. The inventors have also isolated and characterized a cDNA encoding this enzyme. The sequence of the 433-residue BEAT protein does not show high similarity to any previously characterized protein, but has 35-residue region which may be involved in interacting with acetyl CoA. E. coli cells expressing BEAT produced enzymatically active protein, and also synthesized and secreted benzylacetate into the medium.

Levels of BEAT mRNA in different parts of the flower correlated positively with levels of BEAT activity, both being highest in petals. The levels of BEAT mRNA in the petals increased as the bud matured, and peaked at anthesis, paralleling changes in BEAT activity. However, three day after anthesis mRNA levels began a steep decline, whereas BEAT activity remained high for the next two d, suggesting that the BEAT protein is relatively stable. A C. breweri line that had low BEAT activity in petals nonetheless had mRNA levels comparable to those found in the high-activity line. Genetic analysis showed that this polymorphism was a quantitative character, possibly controlled in part by post-transcriptional mechanisms.

The present invention, in one embodiment, provides an isolated BEAT polypeptide (SEQ ID NO: 2). The present invention further provides an isolated nucleic acid encoding a BEAT polypeptide. This isolated nucleic acid may be derived from C. breweri or various monocot or dicot plants. By screening a genomic library with a C. breweri BEAT probe and PCRTM-based amplification of sequences from C. concinna using oligonucleotides specific for the beginning and end of the C. breweri BEAT gene, a number of C. concinna BEAT-related genes were identified. These genes encode proteins having the sequences of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13.

Disclosed herein are methods and compositions for using BEAT polypeptides and nucleotides that encode BEAT. These methods and compositions may involve expression of a DNA sequence encoding a BEAT polypeptide linked operably to a promoter functional in a plant cell. The use of such methods and compositions result in the alteration in a particular fragrance quality, scent or flavor of a plant. In particular, increase in BEAT activity is associated with an increase in benzylacetate in the plant, which is one of the components responsible for the characteristic aroma of stone fruit plants (Caccioni et al., 1995).

With the identification of the BEAT gene as key gene in the benzenoid biosynthesis pathway, a number of different endeavors become possible. For example, one may employ both protein and DNA compositions in the production of transgenic plants with improved characteristics and greater viability. More specifically, it will be possible to engineer plants to exhibit an altered fragrance and flavor by promoting an increase or decrease in benzylacetate content of plants. It also will be possible to determine which domain (s) of BEAT is (are) responsible for acetyl CoA transfer activity. This will involve the production of truncated, deletion, fusion and replacement mutants of BEAT. In this way, new enzymes with unique activities and/or specificities will be produced. Indeed, the present inventors have shown that there is a small region of homology between BEAT and CER2 of Arabidopsis and GLOSSY2 from maize, these regions have been implicated in elongation of cuticular wax molecules (Tacke et al., 1995; Negruk et al., 1996), suggesting that this region in BEAT may be involved in binding or otherwise interacting with acetyl CoA. Thus, it will be possible to alter the residues in this region and modulate the transferase activity.

In yet other examples, the products and by-products of the benzylacetate biosynthetic pathway may be involved in conferring insect attractant properties, thereby facilitating pollination in important crop plants that require insect pollination. The methods and compositions for achieving these and other objectives are detailed herein below.

II. BEAT Polypeptides and Fragments Thereof Thus, according to one aspect of the present invention, the present inventors provide a novel enzyme, BEAT. This molecule will prove useful in a variety of different contexts. For example, BEAT may be used in catalyzing the acetyl CoA transfer reaction in test samples. BEAT also can be used as part of a screening assay to examine reagents for their ability to affect such transfer in vitro. In another embodiment, BEAT will prove useful in standard laboratory procedures as protein marker of known molecular weight predicted as 48.2 and an apparent molecular weight of 58 kD on SDS-PAGE.

In addition to the entire BEAT molecule, the present invention also relates to fragments of the polypeptide that may or may not retain the acetyl transferase (or other) activity. Fragments including the N-terminus of the molecule may be generated by genetic engineering of translation stop sites within the coding region (discussed below). Alternatively, treatment of the BEAT molecule with proteolytic enzymes, known as protease, can produce a variety of N-terminal, C-terminal and internal fragments. Examples of fragments may include contiguous residues of the BEAT sequence given in SEQ ID N0: 2 of 6,7,8,9,10,11,12,13,14,15,16,17,18, 19,20,21,22,23,24,25,30,35,40,45,50,55,60,65,75,80,85,90,95, 100,200, 300,400 or more amino acids in length. These fragments may be purified according to known methods, such as precipitation (e. g., ammonium sulfate), HPLC, ion exchange chromatography, affinity chromatography (including immunoaffinity chromatography) or various size separations (sedimentation, gel electrophoresis, gel filtration).

A. Structural Features of the Polypeptide The inventors have purified the protein encoded by BEAT cDNA. The protein has 433 amino acids, and its sequence does not show high similarity to any previously characterized protein. Further, the inventors have identified and purified a number of

BEAT-related proteins from C. concinna, including BEAT-1, BEAT-2, BEAT-3 and BEAT-4.

BEAT has a small region of similarity with CER2 of Arabidopsis and GLOSSY2 from maize, implicated in C30 to C32 elongation of cuticular wax molecules (Tacke et al., 1995; Negruk et al., 1996), may be involved in binding or otherwise interacting with acetyl CoA. Although both CER2 and GLOSSY2 do not show any extensive similarity to BEAT outside this short (35 residues) region although their overall length is very similar to that of BEAT.

E. coli cells expressing BEAT not only produced enzymatically active protein, but also synthesized benzylacetate and secreted it into the medium. Although the inventors are not aware of any reports concerning the synthesis of benzylalcohol in E. coli, it is evidently present, perhaps only as an intermediate in a pathway. The medium in which the bacteria expressing BEAT grew also contained, in addition to benzylacetate, a few other unidentified volatiles in lesser amounts, e. g., the minor peak at 12 min, which contained an acetyl group as indicated by MS analysis. It appears that the overexpression of BEAT in the bacterial cells caused acetylation of other compounds, that are likely to be structurally similar to benzylalcohol.

The inventors previously have shown that the levels of activity of two enzymes involved in the biosynthesis of scent volatiles in C. breweri, linalool synthase and (iso) eugenol methyltransferase, closely parallel the level of mRNA of their respective genes (LIS and IEMT) from bud formation up to three days after anthesis (Dudareva et al., 1996; Wang et al., 1997). The levels of these enzymes increase as the bud matures, and peak just before or soon after anthesis. However, three days after anthesis enzyme activities (and protein levels, as examined so far only for LIS) remain high or start to decline slowly, whereas mRNA levels begin to decline much faster. These observations are similar to what the inventors have observed in the present study with BEAT activity and mRNA levels. Taken together, these results indicate that high levels of activity of these scent biosynthetic enzyme depend on high

levels of mRNA, but also that these three enzymes are relatively stable and they persist in the cell for several days after their synthesis.

The inventors also have previously examined polymorphism (strictly, dimorphism) for LIS and IEMT activities in wild populations of Clarkia. Since all C. breweri plants examined make similar amounts of linalool, LIS activity and mRNA levels were compared with those in the closely related, nonscented C. concinna (Dudareva et al., 1996). Dimorphism for IEMT was found within C. breweri, with some plants containing little or no IEMT activity and producing no (iso) methyleugenol (Raguso and Pichersky, 1995; Wang et al., 1997). In both cases, it was found that plants that did not exhibit enzyme activity did not have detectable levels of the corresponding mRNA in their flowers.

The polymorphism for BEAT activity levels is different from these two previous cases because it involves a quantitative variation, not presence or absence of activity. Moreover, the basis for this variation does not appear to lie at the transcriptional level. BEAT mRNA and levels in all parts of the flower were similar in both C. breweri lines (FIG. 8), and so were BEAT activity levels, with the exception of the petals. In the"low-activity"line, BEAT activity in petals was only 7.5% (a 13- fold difference) that of the petals in the"high-activity"plants (Table 4). Since petals constitute the bulk of the flower and contain greater than 90% of the total BEAT activity of the flower, a specific, 13-fold reduction in BEAT activity in the petals is significant. How this reduction occurs without a concomitant reduction in mRNA levels is not clear.

The genetic analysis of this polymorphism indicated that the trait was quantitative. Although by grouping the F2 plants into"high","low", and "intermediate"groups in respect to petal BEAT activity levels, a ratio among these groups which was not significantly different from a 1: 2: 1 ratio could be observed, the delineation between the"intermediate"and"high"classes was somewhat arbitrary.

Another basis for identifying this trait as quantitative was the observation that activity

levels among F individuals raged up to 2.5-fold greater than that of the high-activity parent.

DNA blot analysis showed that although all the F2 individuals in the"low activity"group, which is relatively well defined, were homozygous for the BEAT allele derived from the low-activity parent, at least one individual homozygous for the low-activity allele had activity levels placing it in the low end of the intermediate- activity group. And while the four plants with the highest activity levels were homozygous for the BEAT allele of the high-activity parent, plants with activity levels between 375 to 588 fkat/mg FW, spanning the range from the upper end of the intermediate-activity group to the middle of the high-activity levels, were either heterozygous or homozygous for the high-activity allele in no discernible pattern.

These observations clearly indicate that although the level of BEAT activity is mostly determined by the BEAT alleles that the plant carries, other factors are involved. Since the inventors have measured BEAT activity from several flowers from each plant, and in most cases the results varied by no more than 10%, the inventors believe that measurement inaccuracy alone could not explain all the variation not due to BEAT alleles. However, although all plants were grown in the same growth chamber, it is possible that some of this variation is due to micro- environmental differences, such as amount of water, shade, etc.

It also is possible that other loci contribute to the BEAT phenotype. Since the levels of BEAT mRNA of both high-and low-activity parents was very similar a transcriptional regulation mechanism can be ruled out. It should be stressed that this observation does not negate the inventors'previous, generalized observation that high levels of mRNA are necessary for high levels of scent BEAT (and also LIS and IEMT) activity, only that in this case high levels of BEAT mRNA are not sufficient for high BEAT activity. At present, several hypotheses can be invoked to explain this observation. It is possible that the different alleles of BEAT encode proteins with

different specific activities, although in this case the trait may perhaps be expected to segregate as a simple Mendelian character, and it is also not clear why such a difference will be manifested in petals only. Alternatively, they may encode proteins whose stability in petals may differ in different genetic backgrounds, or their respective mRNAs are translated (in petals) with different efficiencies in different genetic backgrounds. These hypotheses remain to be tested.

B. Functional Characteristics of BEAT Flowers of C. breweri emit a strong sweet fragrance that consists of 8 to 12 volatile compounds. These volatiles are derived from two biochemical pathways, one leading to monoterpenoids and the other to the phenylpropanoids-benzenoids (Raguso and Pcihersky, 1995). Three of the components of the latter group are esters, namely, benzylacetate, benylbenzoate and methylsilicate. The ester benzylacetate is a major constituent of the floral scent of Clarkia breweri, and constitutes up to 40% of the total scent output, with the other two esters, benzylbenzoate and methylsalicylate, contributing about 5% each (Raguso and Pichersky, 1995). The inventors have purified the enzyme acetyl CoA: benzylalcohol acetyl transferase (BEAT), which catalyzes the formation of benzylacetate, from a C. breweri line that has high BEAT activity. Further, using a probes C. breweri BEAT probe and the PCRTM-based amplification of sequences from C. concinna using oligonucleotides specific for the beginning and end of the C. breweri BEAT gene, the inventors identified four different genes that encode proteins BEAT-related proteins (SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, respectively). Thus, the inventors show that the genome of C. concinna also contains BEAT genes, that these genes are expressed in both floral and vegetative tissues.

The inventors'investigation concentrated on the biosynthesis of benzylacetate via the action of BEAT. To date, no enzymatic activity capable of converting benzylalcohol to benzylacetate has been reported from plants. The present invention demonstrates the existence of a single enzyme, designated BEAT, which benzylalcohol to yield benzylacetate, with high specificity. Although this enzyme has

some sequence similarity to CER2 of Arabidopsis and GLOSSY2 from maize which may be involved in binding or otherwise interacting with acetyl CoA, there is no other reported enzymes that catalyze the conversion of benzylalcohol to benzylacetate.

The levels of BEAT activity and mRNA in the different floral tissues of C. breweri strongly correlated with the production and emission of these benzylacetate by the same tissues, being highest in petals, followed by stamens, style, and stigma, and absent in the leaf and stem tissue. Moreover, the total BEAT per flower is approximately 10-fold greater than the activity in LIS. These results are similar to those obtained for LIS and IEMT, where strong positive correlation was observed between levels of enzyme activities and emissions of linalool and (iso) methylisoeugenol from floral tissues (Pichersky et al., 1994; Wang et al., 1997).

The inventors examined BEAT expression in different part of the flower and showed that levels of BEAT mRNA were highest in petals, followed by style, sepals, and stamens. Very low levels were observed in the stigma, and none in leaves. These mRNA levels parallel the BEAT activity profile obtained for this line, in which highest activity is found in petals and none in the leaves. In a second line of C. breweri, plants have a 13-fold reduction in BEAT activity in petals compared with the high-activity line (24 flcat/mg FW vs. 317 fkat/mg FW), whereas BEAT activity levels in other parts of the flower are not significantly different. Surprisingly, BEAT mRNA levels in petals in this line were only 16% lower compared with the levels of BEA T mRNA in petals of high-activity plants. The mRNA levels in other parts of the flower were also similar to those found in the high-activity line.

Flowers of C. breweri usually last 5-6 days, even when not pollinated. The steady-state level of BEAT mRNA in petals of the high-activity line were examined during the lifespan of the flower. mRNA was first detected just before the flower opened, and its level was highest at anthesis. After the second day, mRNA levels declined sharply. It should be noted that in the bud, and during the first two days after anthesis, protein and mRNA levels increased in parallel. However, subsequent to day

2 after anthesis, BEAT protein activity continued to increase up to day 5 after anthesis, when it began to decrease. By contrast, BEAT mRNA levels began to decline on day 3 after anthesis.

The consequence of high levels of BEAT activity without concomitant emission of volatile products in older flowers is unclear. It could be that the volatile compounds still are being made by the floral tissues but are being tied into non- volatile compounds. Loughrin et al. (1992) have reported an increase in glycosidically bound scent compounds in tobacco floral tissues as the flowers aged.

Another possibility is that the products are required for additional processes in the flowers other than scent emission. Yet another alternative is that as the flowers age the substrates are diverted into other compartments and are not accessible to the scent biosynthetic pathway.

When the present application refers to the function of BEAT or"wild-type" activity, it is meant that the enzyme has the ability to mediate the acetyl transfer reaction such that benzylalcohol may be converted to benzylacetate. Thus the molecule in question has the ability to acetylate benzylalcohol. However, it also may acetylate other molecules. Determination of which polypeptides possess this activity may be achieved using assays familiar to those of skill in the art.

For example, cell extracts may be assayed for by transfecting cells with the test cDNA in an appropriate vector and then assaying these cells for acetyl transferase activity. Assays of acetyl transferase activity are well known to those of skill in the art and are described in, for example, in Example 1.

C. VariantsofBEAT The present invention contemplates variants of BEAT. In specific embodiments, the present inventors performed a combined search for C. concinna BEAT-related genes, using the screening of a genomic library with a C. breweri BEAT probe and the PCRTM based amplification of sequences from C. concinna using oligonucleotides

specific for the beginning and end of the C. breweri BEAT gene. These studies yielded four different genes which encode proteins having the sequences of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, respectively. Thus, the present invention contemplates and has demonstrated that the techniques of the present invention are useful in isolating BEAT variants from species other than C. breweri.

Additional amino acid sequence variants also are contemplated. Such amino acid sequence variants of the polypeptide can be substitutional, insertional or deletion variants. Deletion variants lack one or more residues of the native protein which are not essential for function or immunogenic activity, and are exemplified by the variants lacking a transmembrane sequence described above. Another common type of deletion variant is one lacking secretory signal sequences or signal sequences directing a protein to bind to a particular part of a cell. Insertional mutants typically involve the addition of material at a non-terminal point in the polypeptide. This may include the insertion of an epitope or simply a single residue. Terminal additions, called fusion proteins, are discussed below.

Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide, such as stability against proteolytic cleavage, without the loss of other functions or properties. Substitutions of this kind preferably are conservative, that is, one amino acid is replaced with one of similar shape and charge. Conservative substitutions are well known in the art and include, for example, the changes of : alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.

The following is a discussion based upon changing of the amino acids of a protein to create an equivalent, or even an improved, second-generation molecule. For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid substitutions can be made in a protein sequence, and its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the DNA sequences of genes without appreciable loss of their biological utility or activity, as discussed below. Table 1 shows the codons that encode particular amino acids.

In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.

Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics (Kyte and Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7) ; serine (-<BR> 0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2) ; glutamate (-<BR> 3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (- 4.5).

It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i. e., still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within 2 is preferred, those which are within 1 are particularly preferred, and those within 0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U. S. Patent 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. As detailed in U. S. Patent 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 + 1); glutamate (+3.0 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 1); alanine (-0.5); histidine *-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).

It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent and immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within 2 is preferred, those that are within +1 are particularly preferred, and those within 0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

Another embodiment for the preparation of polypeptides according to the invention is the use of peptide mimetic. Mimetic are peptide-containing molecules that mimic elements of protein secondary structure. See, for example, Johnson et al., "Peptide Turn Mimetic"in BIOTECHNOLOGYAND PHARMACY, Pezzuto et al., Eds., Chapman and Hall, New York (1993). The underlying rationale behind the use of peptide mimetic is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions, such as those of antibody and antigen. A peptide mimetic is expected to permit molecular interactions similar to the natural molecule. These principles may be used, in conjunction with the principles outline above, to engineer second generation molecules having many of the natural properties of BEAT, but with altered and even improved characteristics.

D. Domain Switching As described in the Examples, the present inventors have identified a novel acetyltransferase from C. breweri and from C. concinna which catalyzes the addition of a acetate group to the benzylalcohol to yield benzyl acetate. There is a reasonable expectation that other homologs, allelic variants and mutants of this gene exist in related species, such as basil, clove, tomato, potato, corn, soybean, oil rapeseed, wheat, barley, rye and others. Upon isolation of these homologs, variants and mutants, and in conjunction with other analyses, certain active or functional domains can be identified. This will provide a starting point for further mutational analysis of the molecule. One way in which this information can be exploited is in"domain switching." Domain switching involves the generation of chimeric molecules using different but, in this case, related polypeptides. By comparing the sequences for BEAT with the other acetyl transferases, and possibly with mutants and allelic variants of these polypeptides, one can make predictions as to the functionally significant regions of these molecules. It then is possible, to switch related domains of these molecules in an effort to determine the criticality of these regions to BEAT function.

Based on the sequence identity, at the amino acid level, it may be inferred that even small changes in the primary sequence of the molecule will affect function.

Further analysis of mutations and their predicted effect on secondary structure will add to this understanding.

It is proposed, therefore, that a series of chimera will be generated. Primarily, the convenience of restriction sites will dictate the precise location of breakpoints. However, generally, one may produce the following types of chimeras. The 5'half of the chimera may be derived from BEAT, while the 3'half of the molecule may be derived from another acetyl transferase. Conversely, the 5'half of the chimera may be derived from an alternative enzyme, while the 3'half of the molecule may be derived from BEAT.

Another approach is to target particular functional regions of molecules. For example it has been shown by the present inventors that the region between amino acids 135 and 169 may be are the putative acetyl CoA binding sites. It is likely that these regions should be conserved, at least to a certain extent given that acetyl CoA binding is a function that the chimera will need to retain. Nonetheless, these residues may constitute regions of the molecule that, spatially, are in close relation to the substrate molecule and, hence, are involved, directly or indirectly, in substrate specificity. Thus, it will be of interest to focus on this region in generating additional chimeras. For example, it may prove desirable to perform random mutagenesis on this region and screen the resultant clones for the desired activity. It may well be preferable to further define the regions involved by creating more subtle chimeras, for example, in which smaller regions of known acetyl transferase activity may be transferred into BEAT. These residues may confer upon the chimera the ability to acetylate key precursors of fragrance production.

E. Fusion Proteins A specialized kind of insertional variant is the fusion protein. This molecule generally has all or a substantial portion of the native molecule, linked at the N-or C- terminus, to all or a portion of a second polypeptide. For example, fusions typically employ leader sequences from other species to permit the recombinant expression of a protein in a heterologous host. Another useful fusion includes the addition of a immunologically active domain, such as an antibody epitope, to facilitate purification of the fusion protein. Inclusion of a cleavage site at or near the fusion junction will facilitate removal of the extraneous polypeptide after purification. Other useful fusions include linking of functional domains, such as active sites from enzymes, glycosylation domains, cellular targeting signals or transmembrane regions. Fusion to a polypeptide that can be used for purification of the substrate-BEAT complex would serve to isolated the substrate for identification and analysis.

F. Purification of Proteins It will be desirable to purify BEAT or variants thereof. Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the cellular milieu to polypeptide and non- polypeptide fractions. Having separated the polypeptide from other proteins, the polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing. A particularly efficient method of purifying peptides is fast protein liquid chromatography or even HPLC.

Certain-aspects of the present invention concern the purification, and in particular embodiments, the substantial purification, of an encoded protein or peptide. The term"purified protein or peptide"as used herein, is intended to refer to a composition, isolatable from other components, wherein the protein or peptide is purified to any degree relative to its naturally-obtainable state. A purified protein or

peptide therefore also refers to a protein or peptide, free from the environment in which it may naturally occur.

Generally,"purified"will refer to a protein or peptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. Where the term "substantially purified"is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the proteins in the composition.

Various methods for quantifying the degree of purification of the protein or peptide will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptides within a fraction by SDS/PAGE analysis. A preferred method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity, herein assessed by a"-fold purification number."The actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the expressed protein or peptide exhibits a detectable activity.

Various techniques suitable for use in protein purification will be well known to those of skill in the art. These include, for example, precipitation with ammonium sulphate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite and affinity chromatography; isoelectric focusing; gel electrophoresis; and combinations of such and other techniques. As is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified protein or peptide.

There is no general requirement that the protein or peptide always be provided in their most purified state. Indeed, it is contemplated that less substantially purified products will have utility in certain embodiments. Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is appreciated that a cation-exchange column chromatography performed utilizing an HPLC apparatus will generally result in a greater"-fold"purification than the same technique utilizing a low pressure chromatography system. Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein.

It is known that the migration of a polypeptide can vary, sometimes significantly, with different conditions of SDS/PAGE (Capaldi et al., 1977). It will therefore be appreciated that under differing electrophoresis conditions, the apparent molecular weights of purified or partially purified expression products may vary.

High Performance Liquid Chromatography (HPLC) is characterized by a very rapid separation with extraordinary resolution of peaks. This is achieved by the use of very fine particles and high pressure to maintain an adequate flow rate. Separation can be accomplished in a matter of minutes, or at most an hour. Moreover, only a very small volume of the sample is needed because the particles are so small and close-packed that the void volume is a very small fraction of the bed volume. Also, the concentration of the sample need not be very great because the bands are so narrow that there is very little dilution of the sample.

Gel chromatography, or molecular sieve chromatography, is a special type of partition chromatography that is based on molecular size. The theory behind gel chromatography is that the column, which is prepared with tiny particles of an inert substance that contain small pores, separates larger molecules from smaller molecules as they pass through or around the pores, depending on their size. As long as the

material of which the particles are made does not adsorb the molecules, the sole factor determining rate of flow is the size. Hence, molecules are eluted from the column in decreasing size, so long as the shape is relatively constant. Gel chromatography is unsurpassed for separating molecules of different size because separation is independent of all other factors such as pH, ionic strength, temperature, etc. There also is virtually no adsorption, less zone spreading and the elution volume is related in a simple matter to molecular weight.

Affinity Chromatography is a chromatographic procedure that relies on the specific affinity between a substance to be isolated and a molecule that it can specifically bind to. This is a receptor-ligand type interaction. The column material is synthesized by covalently coupling one of the binding partners to an insoluble matrix. The column material is then able to specifically adsorb the substance from the solution. Elution occurs by changing the conditions to those in which binding will not occur (alter pH, ionic strength, temperature, etc.).

A particular type of affinity chromatography useful in the purification of carbohydrate containing compounds is lectin affinity chromatography. Lectins are a class of substances that bind to a variety of polysaccharides and glycoproteins.

Lectins are usually coupled to agarose by cyanogen bromide. Conconavalin A coupled to Sepharose was the first material of this sort to be used and has been widely used in the isolation of polysaccharides and glycoproteins other lectins that have been include lentil lectin, wheat germ agglutinin which has been useful in the purification of N-acetyl glucosaminyl residues and Helix pomatia lectin. Lectins themselves are purified using affinity chromatography with carbohydrate ligands. Lactose has been used to purify lectins from castor bean and peanuts; maltose has been useful in extracting lectins from lentils and jack bean; N-acetyl-D galactosamine is used for purifying lectins from soybean; N-acetyl glucosaminyl binds to lectins from wheat germ; D-galactosamine has been used in obtaining lectins from clams and L-fucose will bind to lectins from lotus.

The matrix should be a substance that itself does not adsorb molecules to any significant extent and that has a broad range of chemical, physical and thermal stability. The ligand should be coupled in such a way as to not affect its binding properties. The ligand should also provide relatively tight binding. And it should be possible to elute the substance without destroying the sample or the ligand. One of the most common forms of affinity chromatography is immunoaffinity chromatography. The generation of antibodies that would be suitable for use in accord with the present invention is discussed below.

G. Synthetic Peptides The present invention also describes smaller BEAT-related peptides for use in various embodiments of the present invention. Because of their relatively small size, the peptides of the invention can also be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Stewart and Young, (1984); Tam et al., (1983); Merrifield, (1986); and Barany and Merrifield (1979), each incorporated herein by reference. Short peptide sequences, or libraries of overlapping peptides, usually from about 6 up to about 35 to 50 amino acids, which correspond to the selected regions described herein, can be readily synthesized and then screened in screening assays designed to identify reactive peptides. Alternatively, recombinant DNA technology may be employed wherein a nucleotide sequence which encodes a peptide of the invention is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression.

III. Nucleic Acids The present invention also provides, in another embodiment, DNAs encoding BEAT or fragments thereof. The present invention identifies the gene for the C. breweri BEAT molecule, however, the present invention is not limited in scope to this gene, as one of ordinary skill in the could, using the nucleic acids disclosed herein, readily identify related homologs in various other species (e. g., basil, clove, tomato,

potato, corn, soybean and others). In certain other embodiments, the inventors used a C. breweri BEAT probe and isolated BEAT-related genes from C. concinna (SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19).

In addition, it should be clear that the present invention is not limited to the specific nucleic acids disclosed herein. As discussed below, an"BEAT gene"may contain a variety of different bases and yet still produce a corresponding polypeptides that is functionally indistinguishable, and in some cases structurally, from the C. breweri gene disclosed herein.

Similarly, any reference to a nucleic acid should be read as encompassing a host cell containing that nucleic acid and, in some cases, capable of expressing the product of that nucleic acid. Cells expressing nucleic acids of the present invention may prove useful in the context of screening for agents that induce, repress, inhibit, augment, interfere with, block, abrogate, stimulate or enhance the function of BEAT.

A. Nucleic Acids Encoding BEAT The gene disclosed in SEQ ID NO: 1 is one of the BEAT genes of the present invention. SEQ ID NO: 1 encodes BEAT in C. breweri. The inventors further have identified additional genes that encode BEAT-like proteins in C. concinna (SEQ ID NO: 16, SEQ ID NO: 17; SEQ ID NO: 18 and SEQ ID NO: 19). Nucleic acids according to the present invention may encode an entire BEAT gene, a domain of BEAT that expresses function capable of mediating methyltransferase activity, or any other fragment of the BEAT sequences set forth herein. The nucleic acid may be derived from genomic DNA, i. e., cloned directly from the genome of a particular organism. In preferred embodiments, however, the nucleic acid would comprise complementary DNA (cDNA). Also contemplated is a cDNA plus a natural intron or an intron derived from another gene; such engineered molecules are sometime referred to as"mini-genes."At a minimum, these and other nucleic acids of the present invention may be used as molecular weight standards in, for example, gel electrophoresis.

The term"cDNA"is intended to refer to DNA prepared using messenger RNA (mRNA) as template. The advantage of using a cDNA, as opposed to genomic DNA or DNA polymerized from a genomic, non-or partially-processed RNA template, is that the cDNA primarily contains coding sequences of the corresponding protein.

There may be times when the full or partial genomic sequence is preferred, such as where the non-coding regions are required for optimal expression or where non- coding regions such as introns are to be targeted in an antisense strategy.

It also is contemplated that a given BEAT from a given species may be represented by natural variants that have slightly different nucleic acid sequences but, nonetheless, encode the same protein (see Table 1 below).

As used in this application, the term"a nucleic acid encoding a BEAT"refers to a nucleic acid molecule that has been isolated free of total cellular nucleic acid. In preferred embodiments, the invention concerns a nucleic acid sequence essentially as set forth in SEQ ID NO: 1. The term"as set forth SEQ ID NO: 1"means that the nucleic acid sequence substantially corresponds to a portion of SEQ ID NO: 1. The term"functionally equivalent codon"is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine (Table 1, below), and also refers to codons that encode biologically equivalent amino acids, as discussed in the following pages.

TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAG Phenylalanine Phe F UUC UW Glycine Gly G GGA GGC GGG GGU Histidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

Allowing for the degeneracy of the genetic code, sequences that have at least about 50%, usually at least about 60%, more usually about 70%, most usually about 80%, preferably at least about 90% and most preferably about 95% of nucleotides that are identical to the nucleotides of SEQ ID NO: I will be sequences that are"as set forth in SEQ ID NO: 1." Sequences that are essentially the same as those set forth in SEQ ID NO: 1 may also be functionally defined as sequences that are capable of

hybridizing to a nucleic acid segment containing the complement of SEQ ID NO: 1 under standard conditions.

The DNA segments of the present invention include those encoding biologically functional equivalent BEAT proteins and peptides, as described above.

Such sequences may arise as a consequence of codon redundancy and amino acid functional equivalency that are known to occur naturally within nucleic acid sequences and the proteins thus encoded. Alternatively, functionally equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by man may be introduced through the application of site-directed mutagenesis techniques or may be introduced randomly and screened later for the desired function, as described below.

B. Oligonucleotide Probes and Primers Naturally, the present invention also encompasses DNA segments that are complementary, or essentially complementary, to the sequence set forth in SEQ ID NO: 1. Nucleic acid sequences that are"complementary"are those that are capable of base-pairing according to the standard Watson-Crick complementary rules. As used herein, the term"complementary sequences"means nucleic acid sequences that are substantially complementary, as may be assessed by the same nucleotide comparison set forth above, or as defined as being capable of hybridizing to the nucleic acid segment of SEQ ID NO: 1 under relatively stringent conditions such as those described herein. Such sequences may encode the entire BEAT protein or functional or non- functional fragments thereof.

Alternatively, the hybridizing segments may be shorter oligonucleotides.

Sequences of 17 bases long should occur only once in the genome and, therefore, suffice to specify a unique target sequence. Although shorter oligomers are easier to make and increase in vivo accessibility, numerous other factors are involved in

determining the specificity of hybridization. Both binding affinity and sequence specificity of an oligonucleotide to its complementary target increases with increasing length. It is contemplated that exemplary oligonucleotides of 8,9,10,11,12,13,14, 15,16,17,18,19,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90, 95,100 or more base pairs will be used, although others are contemplated. Longer polynucleotides encoding 250,500,1000,1212,1500, or 1564 bases and longer are contemplated as well. Such oligonucleotides will find use, for example, as probes in Southern and Northern blots and as primers in amplification reactions.

Suitable hybridization conditions will be well known to those of skill in the art. In certain applications, for example, substitution of amino acids by site-directed mutagenesis, it is appreciated that lower stringency conditions are required. Under these conditions, hybridization may occur even though the sequences of probe and target strand are not perfectly complementary, but are mismatched at one or more positions. Conditions may be rendered less stringent by increasing salt concentration and decreasing temperature. For example, a medium stringency condition could be provided by about 0.1 to 0.25 M NaCI at temperatures of about 37°C to about 55°C, while a low stringency condition could be provided by about 0.15 M to about 0.9 M salt, at temperatures ranging from about 20°C to about 55°C. Thus, hybridization conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results.

One method of using probes and primers of the present invention is in the search for genes related to BEAT or, more particularly, homologs of BEAT from other species.

Normally, the target DNA will be a genomic or cDNA library, although screening may involve analysis of RNA molecules. By varying the stringency of hybridization, and the region of the probe, different degrees of homology may be discovered.

Another way of exploiting probes and primers of the present invention is in site-directed, or site-specific mutagenesis. Site-specific mutagenesis is a technique useful in the preparation of individual peptides, or biologically functional equivalent

proteins or peptides, through specific mutagenesis of the underlying DNA. The technique further provides a ready ability to prepare and test sequence variants, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA. Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Typically, a primer of about 17 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered.

The technique typically employs a bacteriophage vector that exists in both a single-stranded and double-stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the M 13 phage. These phage vectors are commercially available and their use is generally well known to those skilled in the art. Double stranded plasmids are also routinely employed in site directed mutagenesis, which eliminates the step of transferring the gene of interest from a phage to a plasmid.

In general, site-directed mutagenesis is performed by first obtaining a single- stranded vector, or melting of two strands of a double stranded vector which includes within its sequence a DNA sequence encoding the desired protein. An oligonucleotide primer bearing the desired mutated sequence is synthetically prepared. This primer is then annealed with the single-stranded DNA preparation, taking into account the degree of mismatch when selecting hybridization conditions, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected that include recombinant vectors bearing the mutated sequence arrangement.

The preparation of sequence variants of the selected gene using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting, as there are other ways in which sequence variants of genes may be obtained. For example, recombinant vectors encoding the desired gene may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.

C. Antisense Constructs Antisense treatments are one way of inhibiting lignin biosynthesis in a plant.

Antisense technology may be used to"knock-out"the function of the BEAT gene or other related biosynthetic genes, thereby decreasing or eliminating the production of benzylacetate and related compounds in a transformed plant cell or whole plant.

Antisense methodology takes advantage of the fact that nucleic acids tend to pair with"complementary"sequences. By complementary, it is meant that polynucleotides are those which are capable of base-pairing according to the standard Watson-Crick complementarity rules. That is, the larger purines will base pair with the smaller pyrimidines to form combinations of guanine paired with cytosine (G: C) and adenine paired with either thymine (A: T) in the case of DNA, or adenine paired with uracil (A: U) in the case of RNA. Inclusion of less common bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others in hybridizing sequences does not interfere with pairing.

Targeting double-stranded (ds) DNA with polynucleotides leads to triple-helix formation; targeting RNA will lead to double-helix formation. Antisense polynucleotides, when introduced into a target cell, specifically bind to their target polynucleotide and interfere with transcription, RNA processing, transport, translation and/or stability. Antisense RNA constructs, or DNA encoding such antisense RNA's, may be employed to inhibit gene transcription or translation or both.

Antisense constructs may be designed to bind to the promoter and other control regions, exons, introns or even exon-intron boundaries of a gene. It is contemplated that the most effective antisense constructs will include regions complementary to intron/exon splice junctions. Thus, it is proposed that a preferred embodiment includes an antisense construct with complementarity to regions within 50-200 bases of an intron-exon splice junction. It has been observed that some exon sequences can be included in the construct without seriously affecting the target selectivity thereof. The amount of exonic material included will vary depending on the particular exon and intron sequences used. One can readily test whether too much exon DNA is included simply by testing the constructs in vitro to determine whether normal cellular function is affected or whether the expression of related genes having complementary sequences is affected.

As stated above,"complementary"or"antisense"means polynucleotide sequences that are substantially complementary over their entire length and have very few base mismatches. For example, sequences of fifteen bases in length may be termed complementary when they have complementary nucleotides at thirteen or fourteen positions. Naturally, sequences which are completely complementary will be sequences which are entirely complementary throughout their entire length and have no base mismatches. Other sequences with lower degrees of homology also are contemplated. For example, an antisense construct which has limited regions of high homology, but also contains a non-homologous region (e. g., ribozyme; see below) could be designed. These molecules, though having less than 50% homology, would bind to target sequences under appropriate conditions.

It may be advantageous to combine portions of genomic DNA with cDNA or synthetic sequences to generate specific constructs. For example, where an intron is desired in the ultimate construct, a genomic clone will need to be used. The cDNA or a synthesized polynucleotide may provide more convenient restriction sites for the remaining portion of the construct and, therefore, would be used for the rest of the sequence.

IV. Generating Antibodies Reactive With BEAT Proteins In another aspect, the present invention contemplates an antibody that is immunoreactive with a BEAT molecule of the present invention, or any portion thereof. An antibody can be a polyclonal or a monoclonal antibody. In a preferred embodiment, an antibody is a monoclonal antibody. Means for preparing and characterizing antibodies are well known in the art (see, e. g., Howell and Lane, 1988).

In a particular embodiment, one may exploit antibodies to BEAT or variants thereof as a way of distinguishing various chimeric constructs. In particular, antibodies that recognize epitopes on these antigens that are lost or disturbed in chimeras may be used to identify BEAT polypeptides while not reacting with the chimeras. Thus, these antibodies will prove useful in examining expression levels of cells and in selection of cells where the cells express a BEAT.

Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogen comprising a polypeptide of the present invention and collecting antisera from that immunized animal. A wide range of animal species can be used for the production of antisera. Typically an animal used for production of anti-antisera is a non-human animal including rabbits, mice, rats, hamsters, pigs or horses. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.

Antibodies, both polyclonal and monoclonal, specific for isoforms of antigen may be prepared using conventional immunization techniques, as will be generally known to those of skill in the art. A composition containing antigenic epitopes of the compounds of the present invention can be used to immunize one or more experimental animals, such as a rabbit or mouse, which will then proceed to produce specific antibodies against the compounds of the present invention. Polyclonal

antisera may be obtained, after allowing time for antibody generation, simply by bleeding the animal and preparing serum samples from the whole blood.

It is proposed that the monoclonal antibodies of the present invention will find useful application in standard immunochemical procedures, such as ELISA and Western blot methods and in immunohistochemical procedures such as tissue staining, as well as in other procedures which may utilize antibodies specific to BEAT-related antigen epitopes. Additionally, it is proposed that monoclonal antibodies specific to a particular BEAT protein of different species may be utilized in other useful applications.

In general, both polyclonal and monoclonal antibodies against BEAT may be used in a variety of embodiments. For example, they may be employed in antibody cloning protocols to obtain cDNAs or genes encoding other BEAT proteins. They may also be used in inhibition studies to analyze the effects of BEAT related peptides in cells or plants. Anti-BEAT antibodies will also be useful in immunolocalization studies to analyze the distribution of BEAT during various cellular events or stages of development. A particularly useful application of such antibodies is in purifying native or recombinant BEAT polypeptides, for example, using an antibody affinity column. The operation of all such immunological techniques will be known to those of skill in the art in light of the present disclosure.

Means for preparing and characterizing antibodies are well known in the art (see, e. g., Harlow and Lane, 1988; incorporated herein by reference). More specific examples of monoclonal antibody preparation are given in the examples below.

As is well known in the art, a given composition may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier.

Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or

rabbit serum albumin can also be used as carriers. Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine.

As also is well known in the art, the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Exemplary and preferred adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant.

The amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster, injection may also be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate mAbs. mAbs may be readily prepared through use of well-known techniques, such as those exemplified in U. S. Patent 4,196,265, incorporated herein by reference. Typically, this technique involves immunizing a suitable animal with a selected immunogen composition, e. g., a purified or partially purified BEAT protein, polypeptide or peptide or cell expressing high levels of BEAT. The immunizing composition is administered in a manner effective to stimulate antibody producing cells. Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep frog cells is also possible. The use of rats may provide certain advantages

(Goding, 1986), but mice are preferred, with the BALB/c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions.

Following immunization, somatic cells with the potential for producing antibodies, specifically B-lymphocytes (B-cells), are selected for use in the mAb generating protocol. These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible. Often, a panel of animals will have been immunized and the spleen of animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe. Typically, a spleen from an immunized mouse contains approximately 5 x 107 to 2 x 108 lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas).

Any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, 1986; Campbell, 1984). For example, where the immunized animal is a mouse, one may use P3-X63/Ag8, P3-X63-Ag8.653, NSl/l. Ag 4 1, Sp210-Agl4, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XXO Bul; for rats, one may use R210. RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with cell fusions.

Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2: 1 ratio, though the ratio may vary from about 20: 1 to about 1: 1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Sendai virus have been described (Kohler and Milstein, 1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by Gefter et al., (1977). The use of electrically induced fusion methods is also appropriate (Goding, 1986).

Fusion procedures usually produce viable hybrids at low frequencies, around 1 x 10-6 to 1 x 10-8. However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, unfused cells (particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium. The selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e. g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B-cells can operate this pathway, but they have a limited life span in culture and generally die within about two wk.. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B-cells.

This culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three wk) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.

The selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide mAbs. The cell lines may be exploited for mAb production in two basic ways. A sample of the hybridoma can be injecte (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion. The injecte animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide mAbs in high concentration. The individual cell lines could also be cultured in vitro, where the mAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations. mAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.

V. Genetic Analysis of BEAT Transgenic Plants One embodiment of the instant invention comprises a method for detecting variation in the expression of BEAT genes. As used herein, the term"BEAT gene"is meant to represent a gene of benzylacetate biosynthesis which includes BEAT, and other acyl transferases capable of the transfer of acetyl group to a benyzlalcohol derivative in the benzylacetate biosynthetic pathway. This method may comprise determining that level of BEAT protein or determining specific alterations in the expressed product. Obviously, this sort of assay has importance in the screening of transformants for potential changes in flavor, fragrance and scent production. Such

assays may in some cases be faster, more accurate or less expensive than conventional assays.

The biological sample may potentially be any type of plant tissue. Nucleic acid is isolated from cells contained in the biological sample, according to standard methodologies (Sambrook et al., 1989). The nucleic acid may be genomic DNA or fractionated or whole cell RNA. Where RNA is used, it may be desired to convert the RNA to a complementary DNA. In one embodiment, the RNA is whole cell RNA; in another, it is poly-A RNA. Normally, the nucleic acid is amplified.

Depending on the format, the specific nucleic acid of interest is identified in the sample directly using amplification or with a second, known nucleic acid following amplification. Next, the identified product is detected. In certain applications, the detection may be performed by visual means (e. g., ethidium bromide staining of a gel). Alternatively, the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of radiolabel or fluorescent label or even via a system using electrical or thermal impulse signals (Affymax Technology; Bellus, 1994).

Following detection, one may compare the results seen in a given plant with a statistically significant reference group of non-transformed control plants. Typically, the non-transformed control plants will be of a genetic background similar to the transformed plants. In this way, it is possible to detect differences in the amount or kind of BEAT protein detected in various transformed plants.

A variety of different assays are contemplated in the screening of plants for particular BEAT transgenes and associated exogenous elements. These techniques may in cases be used to detect for both the presence of the particular genes as well as rearrangements that may have occurred in the gene construct. The techniques include but are not limited to, fluorescent in situ hybridization (FISH), direct DNA sequencing, PFGE analysis, Southern or Northern blotting, single-stranded

conformation analysis (SSCA), RNAse protection assay, allele-specific oligonucleotide (ASO), dot blot analysis, denaturing gradient gel electrophoresis, RFLP and PCRTM-SSCP.

A. Primers and Probes The term primer, as defined herein, is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template- dependent process. Typically, primers are oligonucleotides from ten to twenty base pairs in length, but longer sequences can be employed. Primers may be provided in double-stranded or single-stranded form, although the single-stranded form is preferred. Probes are defined differently, although they may act as primers. Probes, while perhaps capable of priming, are designed to binding to the target DNA or RNA and need not be used in an amplification process.

In preferred embodiments, the probes or primers are labeled with radioactive species (32p, 14C, 3sS, 3H, or other label), with a fluorophore (rhodamine, fluorescein), an antigen (biotin, streptavidin, digoxigenin), or a chemillumiscent (luciferase).

B. Template Dependent Amplification Methods A number of template dependent processes are available to amplify the marker sequences present in a given template sample. One of the best known amplification methods is the polymerase chain reaction (referred to as PCR) which is described in detail in U. S. Patent Nos. 4,683,195,4,683,202 and 4,800,159, and in Innis et al., 1990, each of which is incorporated herein by reference in its entirety.

Briefly, in PCRTM, two primer sequences are prepared that are complementary to regions on opposite complementary strands of the marker sequence. An excess of deoxynucleoside triphosphates are added to a reaction mixture along with a DNA polymerase, e. g., Taq polymerase. If the marker sequence is present in a sample, the primers will bind to the marker and the polymerase will cause the primers to be extended along the marker sequence by adding on nucleotides. By raising and

lowering the temperature of the reaction mixture, the extended primers will dissociate from the marker to form reaction products, excess primers will bind to the marker and to the reaction products and the process is repeated.

A reverse transcriptase PCRz amplification procedure may be performed in order to quantify the amount of mRNA amplified. Methods of reverse transcribing RNA into cDNA are well known and described in Sambrook et al., 1989. Alternative methods for reverse transcription utilize thermostable, RNA-dependent DNA polymerases. These methods are described in WO 90/07641 filed December 21, 1990. Polymerase chain reaction methodologies are well known in the art.

Another method for amplification is the ligase chain reaction ("LCR"), disclosed in EPO No. 320 308, incorporated herein by reference in its entirety. In LCR, two complementary probe pairs are prepared, and in the presence of the target sequence, each pair will bind to opposite complementary strands of the target such that they abut. In the presence of a ligase, the two probe pairs will link to form a single unit. By temperature cycling, as in PCRTM, bound ligated units dissociate from the target and then serve as"target sequences"for ligation of excess probe pairs. U. S.

Patent 4,883,750 describes a method similar to LCR for binding probe pairs to a target sequence.

Qbeta Replicase, described in PCT Application No. PCT/US87/00880, may also be used as still another amplification method in the present invention. In this method, a replicative sequence of RNA that has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase. The polymerase will copy the replicative sequence that can then be detected.

An isothermal amplification method, in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5'- [a-thio]-triphosphates in one strand of a restriction site may also be

useful in the amplification of nucleic acids in the present invention, Walker et al., (1992).

Strand Displacement Amplification (SDA) is another method of carrying out isothermal amplification of nucleic acids which involves multiple rounds of strand displacement and synthesis, i. e., nick translation. A similar method, called Repair Chain Reaction (RCR), involves annealing several probes throughout a region targeted for amplification, followed by a repair reaction in which only two of the four bases are present. The other two bases can be added as biotinylated derivatives for easy detection. A similar approach is used in SDA. Target specific sequences can also be detected using a cyclic probe reaction (CPR). In CPR, a probe having 3'and 5'sequences of non-specific DNA and a middle sequence of specific RNA is hybridized to DNA that is present in a sample. Upon hybridization, the reaction is treated with RNase H, and the products of the probe identified as distinctive products that are released after digestion. The original template is annealed to another cycling probe and the reaction is repeated.

Still another amplification methods described in GB Application No. 2 202 328, and in PCT Application No. PCT/US89/01025, each of which is incorporated herein by reference in its entirety, may be used in accordance with the present invention. In the former application,"modified"primers are used in a PCRTM-like, template-and enzyme-dependent synthesis. The primers may be modified by labeling with a capture moiety (e. g., biotin) and/or a detector moiety (e. g., enzyme). In the latter application, an excess of labeled probes are added to a sample. In the presence of the target sequence, the probe binds and is cleaved catalytically. After cleavage, the target sequence is released intact to be bound by excess probe. Cleavage of the labeled probe signals the presence of the target sequence.

Other nucleic acid amplification procedures include transcription-based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR (Kwoh et al., 1989; Gingeras et al., PCT Application WO

88/10315, incorporated herein by reference in their entirety). In NASBA, the nucleic acids can be prepared for amplification by standard phenol/chloroform extraction, heat denaturation of a clinical sample, treatment with lysis buffer and minispin columns for isolation of DNA and RNA or guanidinium chloride extraction of RNA. These amplification techniques involve annealing a primer which has target specific sequences. Following polymerization, DNA/RNA hybrids are digested with RNase H while double stranded DNA molecules are heat denatured again. In either case the single stranded DNA is made fully double stranded by addition of second target specific primer, followed by polymerization. The double-stranded DNA molecules are then multiply transcribed by an RNA polymerase such as T7 or SP6. In an isothermal cyclic reaction, the RNA's are reverse transcribed into single stranded DNA, which is then converted to double stranded DNA, and then transcribed once again with an RNA polymerase such as T7 or SP6. The resulting products, whether truncated or complete, indicate target specific sequences.

Davey et al., EPO No. 329 822 (incorporated herein by reference in its entirety) disclose a nucleic acid amplification process involving cyclically synthesizing single-stranded RNA ("ssRNA"), ssDNA, and double-stranded DNA (dsDNA), which may be used in accordance with the present invention. The ssRNA is a template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase). The RNA is then removed from the resulting DNA: RNA duplex by the action of ribonuclease H (RNase H, an RNase specific for RNA in duplex with either DNA or RNA). The resultant ssDNA is a template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5'to its homology to the template. This primer is then extended by DNA polymerase (exemplified by the large "Klenow"fragment of E. coli DNA polymerase I), resulting in a double-stranded DNA ("dsDNA") molecule, having a sequence identical to that of the original RNA between the primers and having additionally, at one end, a promoter sequence. This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then re-enter the cycle leading to very

swift amplification. With proper choice of enzymes, this amplification can be done isothermally without addition of enzymes at each cycle. Because of the cyclical nature of this process, the starting sequence can be chosen to be in the form of either DNA or RNA.

Miller et al., PCT Application WO 89/06700 (incorporated herein by reference in its entirety) disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter/primer sequence to a target single-stranded DNA ("ssDNA") followed by transcription of many RNA copies of the sequence. This scheme is not cyclic, i. e., new templates are not produced from the resultant RNA transcripts. Other amplification methods include"RACE"and"one-sided PCR" (Frohman, M. A., In: PCRTM PROTOCOLS. A GUIDE TO METHODS AND APPLICATIONS, Academic Press, N. Y., 1990; Ohara et al., 1989; each herein incorporated by reference in their entirety).

Methods based on ligation of two (or more) oligonucleotides in the presence of nucleic acid having the sequence of the resulting"di-oligonucleotide", thereby amplifying the di-oligonucleotide, may also be used in the amplification step of the present invention. Wu et al., (1989), incorporated herein by reference in its entirety.

C. Southern/Northern Blotting Blotting techniques are well known to those of skill in the art. Southern blotting involves the use of DNA as a target, whereas Northern blotting involves the use of RNA as a target. Each provide different types of information, although cDNA blotting is analogous, in many aspects, to blotting or RNA species.

Briefly, a probe is used to target a DNA or RNA species that has been immobilized on a suitable matrix, often a filter of nitrocellulose. The different species should be spatially separated to facilitate analysis. This often is accomplished by gel electrophoresis of nucleic acid species followed by"blotting"on to the filter.

Subsequently, the blotted target is incubated with a probe (usually labeled) under conditions that promote denaturation and rehybridization. Because the probe is designed to base pair with the target, the probe will binding a portion of the target sequence under renaturing conditions. Unbound probe is then removed, and detection is accomplished as described above.

D. Separation Methods It normally is desirable, at one stage or another, to separate the amplification product from the template and the excess primer for the purpose of determining whether specific amplification has occurred. In one embodiment, amplification products are separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods. See Sambrook et al., 1989.

Alternatively, chromatographic techniques may be employed to effect separation. There are many kinds of chromatography which may be used in the present invention: adsorption, partition, ion-exchange and molecular sieve, and many specialized techniques for using them including column, paper, thin-layer and gas chromatography (Freifelder, 1982).

E. Detection Methods Products may be visualized in order to confirm amplification of the marker sequences. One typical visualization method involves staining of a gel with ethidium bromide and visualization under UV light. Alternatively, if the amplification products are integrally labeled with radio-or fluorometrically-labeled nucleotides, the amplification products can then be exposed to x-ray film or visualized under the appropriate stimulating spectra, following separation.

In one embodiment, visualization is achieved indirectly. Following separation of amplification products, a labeled nucleic acid probe is brought into contact with the amplified marker sequence. The probe preferably is conjugated to a chromophore but may be radiolabeled. In another embodiment, the probe is conjugated to a binding

partner, such as an antibody or biotin, and the other member of the binding pair carries a detectable moiety.

In one embodiment, detection is by a labeled probe. The techniques involved are well known to those of skill in the art and can be found in many standard books on molecular protocols. See Sambrook et al., 1989. For example, chromophore or radiolabel probes or primers identify the target during or following amplification.

One example of the foregoing is described in U. S. Patent No. 5,279,721, incorporated by reference herein, which discloses an apparatus and method for the automated electrophoresis and transfer of nucleic acids. The apparatus permits electrophoresis and blotting without external manipulation of the gel and is ideally suited to carrying out methods according to the present invention.

In addition, the amplification products described above may be subjected to sequence analysis to identify specific kinds of variations using standard sequence analysis techniques. Within certain methods, exhaustive analysis of genes is carried out by sequence analysis using primer sets designed for optimal sequencing (Pignon et al, 1994). The present invention provides methods by which any or all of these types of analyses may be used. Using the sequences disclosed herein, oligonucleotide primers may be designed to permit the amplification of sequences throughout the BEAT or other BEAT genes that may then be analyzed by direct sequencing.

F. Kit Components All the essential materials and reagents required for detecting and sequencing BEAT or other BEAT genes, and variants thereof may be assembled together in a kit.

This generally will comprise preselected primers and probes. Also included may be enzymes suitable for amplifying nucleic acids including various polymerases (RT, Taq, SequenaseTM etc.), deoxynucleotides and buffers to provide the necessary reaction mixture for amplification. Such kits also generally will comprise, in suitable

means, distinct containers for each individual reagent and enzyme as well as for each primer or probe.

G. Design and Theoretical Considerations for Relative Quantitative RT-PCR Reverse transcription (RT) of RNA to cDNA followed by relative quantitative PCR (RT-PCR3 can be used to determine the relative concentrations of specific mRNA species isolated from plants. By determining that the concentration of a specific mRNA species varies, it is shown that the gene encoding the specific mRNA species is differentially expressed.

In PCR, the number of molecules of the amplified target DNA increase by a factor approaching two with every cycle of the reaction until some reagent becomes limiting. Thereafter, the rate of amplification becomes increasingly diminished until there is no increase in the amplified target between cycles. If a graph is plotted in which the cycle number is on the X axis and the log of the concentration of the amplified target DNA is on the Y axis, a curved line of characteristic shape is formed by connecting the plotted points. Beginning with the first cycle, the slope of the line is positive and constant. This is said to be the linear portion of the curve. After a reagent becomes limiting, the slope of the line begins to decrease and eventually becomes zero. At this point the concentration of the amplified target DNA becomes asymptotic to some fixed value. This is said to be the plateau portion of the curve.

The concentration of the target DNA in the linear portion of the PCR TM amplification is directly proportional to the starting concentration of the target before the reaction began. By determining the concentration of the amplified products of the target DNA in PCR reactions that have completed the same number of cycles and are in their linear ranges, it is possible to determine the relative concentrations of the specific target sequence in the original DNA mixture. If the DNA mixtures are cDNAs synthesized from RNAs isolated from different tissues or cells, the relative abundances of the specific mRNA from which the target sequence was derived can be

determined for the respective tissues or cells. This direct proportionality between the concentration of the PCRTM products and the relative mRNA abundances is only true in the linear range of the PCRTM reaction.

The final concentration of the target DNA in the plateau portion of the curve is determined by the availability of reagents in the reaction mix and is independent of the original concentration of target DNA. Therefore, the first condition that must be met before the relative abundances of a mRNA species can be determined by RT- PCRTM for a collection of RNA populations is that the concentrations of the amplified PCRTM products must be sampled when the PCR Tm reactions are in the linear portion of their curves.

The second condition that must be met for an RT-PCR study to successfully determine the relative abundances of a particular mRNA species is that relative concentrations of the amplifiable cDNAs must be normalized to some independent standard. The goal of an RT-PCRTM study is to determine the abundance of a particular mRNA species relative to the average abundance of all mRNA species in the sample.

Most protocols for competitive PCRTM utilize internal PCRTM standards that are approximately as abundant as the target. These strategies are effective if the products of the PCRTM amplifications are sampled during their linear phases. If the products are sampled when the reactions are approaching the plateau phase, then the less abundant product becomes relatively over represented. Comparisons of relative abundances made for many different RNA samples, such as is the case when examining RNA samples for differential expression, become distorted in such a way as to make differences in relative abundances of RNAs appear less than they actually are. This is not a significant problem if the internal standard is much more abundant than the target. If the internal standard is more abundant than the target, then direct linear comparisons can be made between RNA samples.

The above discussion describes theoretical considerations for an RT-PCR assay for plant tissue. The problems inherent in plant tissue samples are that they are of variable quantity (making normalization problematic), and that they are of variable quality (necessitating the co-amplification of a reliable internal control, preferably of larger size than the target). Both of these problems are overcome if the RT-PCR is performed as a relative quantitative RT-PCR with an internal standard in which the internal standard is an amplifiable cDNA fragment that is larger than the target cDNA fragment and in which the abundance of the mRNA encoding the internal standard is roughly 5-100 fold higher than the mRNA encoding the target. This assay measures relative abundance, not absolute abundance of the respective mRNA species.

Other studies may be performed using a more conventional relative quantitative RT-PCRz assay with an external standard protocol. These assays sample the PCRTM products in the linear portion of their amplification curves. The number of PCRTM cycles that are optimal for sampling must be empirically determined for each target cDNA fragment. In addition, the reverse transcriptase products of each RNA population isolated from the various tissue samples must be carefully normalized for equal concentrations of amplifiable cDNAs. This consideration is very important since the assay measures absolute mRNA abundance.

Absolute mRNA abundance can be used as a measure of differential gene expression only in normalized samples. While empirical determination of the linear range of the amplification curve and normalization of cDNA preparations are tedious and time consuming processes, the resulting RT-PCR assays can be superior to those derived from the relative quantitative RT-PCR assay with an internal standard.

One reason for this advantage is that without the internal standard/competitor, all of the reagents can be converted into a single PCRT product in the linear range of the amplification curve, thus increasing the sensitivity of the assay. Another reason is that with only one PCRTM product, display of the product on an electrophoretic gel or another display method becomes less complex, has less background and is easier to interpret.

H. Chip Technologies Specifically contemplated by the present inventors are chip-based DNA technologies such as those described by Hacia et al. (1996) and Shoemaker et al.

(1996). Briefly, these techniques involve quantitative methods for analyzing large numbers of genes rapidly and accurately. By tagging genes with oligonucleotides or using fixed probe arrays, one can employ chip technology to segregate target molecules as high density arrays and screen these molecules on the basis of hybridization. See also Pease et al. (1994); Fodor et al. (1991).

VI. Immunoassays for BEAT Gene Expression Antibodies of the present invention can be used in characterizing the expression of BEAT genes, through techniques such as ELISAs and Western blotting.

This may provide a more efficient, accurate or cost effective method for screening plants as part of a standard breeding program or that have been transformed with BEAT genes for relative rates of phenylpropanoid biosynthesis, aiding in the selection of plant products that have a reduced clove-like flavor.

The use of antibodies of the present invention, in an ELISA assay is contemplated. For example, anti-BEAT proteins are immobilized onto a selected surface, preferably a surface exhibiting a protein affinity such as the wells of a polystyrene microtiter plate. After washing to remove incompletely adsorbed material, it is desirable to bind or coat the assay plate wells with a non-specific protein that is known to be antigenically neutral with regard to the test antisera such as bovine serum albumin (BSA), casein or solutions of powdered milk. This allows for blocking of non-specific adsorption sites on the immobilizing surface and thus reduces the background caused by non-specific binding of antigen onto the surface.

After binding of antibody to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing

surface is contacted with the sample to be tested in a manner conducive to immune complex (antigen/antibody) formation.

Following formation of specific immunocomplexes between the test sample and the bound antibody, and subsequent washing, the occurrence and even amount of immunocomplex formation may be determined by subjecting same to a second antibody having specificity for BEAT proteins that differs from the first antibody.

Appropriate conditions preferably include diluting the sample with diluents such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween.

These added agents also tend to assist in the reduction of nonspecific background.

The layered antisera is then allowed to incubate for from about 2 to about 4 hours, at temperatures preferably on the order of about 25° to about 27°C. Following incubation, the antisera-contacted surface is washed so as to remove non- immunocomplexed material. A preferred washing procedure includes washing with a solution such as PBS/Tween, or borate buffer.

To provide a detecting means, the second antibody will preferably have an associated enzyme that will generate a color development upon incubating with an appropriate chromogenic substrate. Thus, for example, one will desire to contact and incubate the second antibody-bound surface with a urease or peroxidase-conjugated anti-human IgG for a period of time and under conditions which favor the development of immunocomplex formation (e. g., incubation for 2 hour at room temperature in a PBS-containing solution such as PBS/Tween).

After incubation with the second enzyme-tagged antibody, and subsequent to washing to remove unbound material, the amount of label is quantified by incubation with a chromogenic substrate such as urea and bromocresol purple or 2,2'-azino-di- (3- ethyl-benzthiazoline)-6-sulfonic acid (ABTS) and H202, in the case of peroxidase as the enzyme label. Quantitation is then achieved by measuring the degree of color generation, e. g., using a visible spectrum spectrophotometer.

The preceding format may be altered by first binding the sample to the assay plate. Then, primary antibody is incubated with the assay plate, followed by detecting of bound primary antibody using a labeled second antibody with specificity for the primary antibody.

VII. Expression Vectors The present invention contemplates an expression vector comprising a polynucleotide of the present invention. Thus, in one embodiment an expression vector is an isolated and purified DNA molecule comprising a promoter operatively linked to a coding region that encodes a polypeptide of the present invention, which coding region is operatively linked to a transcription-terminating region, whereby the promoter drives the transcription of the coding region.

As used herein, the term"operatively linked"means that a promoter is connected to an coding region in such a way that the transcription of that coding region is controlled and regulated by that promoter. Means for operatively linking a promoter to a coding region are well known in the art.

In a preferred embodiment, the recombinant expression of DNAs encoding the BEAT proteins of the present invention is preferable in gram-negative bacterium such as an E. coli or Pseudomonas spp. host cell. Promoters which function in high-level expression of target polypeptides in E. coli and other Gram-negative host cells are well-known in the art. Alternatively, mutagenized or recombinant BEAT protein- encoding gene promoters may be engineered by the hand of man and used to promote expression of the novel gene segments disclosed herein.

Where an expression vector of the present invention is to be used to transform a plant, a promoter is selected that has the ability to drive expression in plants. Promoters that function in plants are also well known in the art. Useful in expressing the polypeptide in plants are promoters that are inducible, viral, synthetic, constitutive

as described (Poszkowski et al., 1989; Odell et al., 1985), and temporally regulated, spatially regulated, and spatio-temporally regulated (Chau et al., 1989).

A promoter is also selected for its ability to direct the transformed plant cell's or transgenic plant's transcriptional activity to the coding region. Structural genes can be driven by a variety of promoters in plant tissues. Promoters can be near- constitutive, such as the CaMV 35S promoter, actin promoter (U. S. Patent 5,641,876 incorporated herein by reference), histone promoters (U. S. Patent 5,491,288 ; U. S.

Patent 5,792,930, each incorporated herein by reference) and ubiquitin promoters (U. S. Patent 5,614,399; U. S. Patent 5,510,474 each incorporated herein by reference) or tissue-specific or developmentally specific promoters affecting dicots or monocots.

Where the promoter is a near-constitutive promoter such as CaMV 35S, increases in polypeptide expression are found in a variety of transformed plant tissues (e. g, callus, leaf, seed and root). Alternatively, the effects of transformation can be directed to specific plant tissues by using plant integrating vectors containing a tissue- specific promoter.

An exemplary tissue-specific promoter is the lectin promoter, which is specific for seed tissue. The Lectin protein in soybean seeds is encoded by a single gene (Lel) that is only expressed during seed maturation and accounts for about 2 to about 5% of total seed mRNA. The lectin gene and seed-specific promoter have been fully characterized and used to direct seed specific expression in transgenic tobacco plants (Vodkin et al., 1983; Lindstrom et al., 1990.).

An expression vector containing a coding region that encodes a polypeptide of interest is engineered to be under control of the lectin promoter and that vector is introduced into plants using, for example, a protoplast transformation method (Dhir et al., 1991; U. S. Patent 5,231,019; U. S. Patent 4,743,548; U. S. Patent 5,272,072; U. S.

Patent 5,591,616; U. S. Patent 5,792,935; U. S. Patent 5,770,450; U. S. Patent 5,597,945; U. S. Patent 5,750,870; U. S. Patent 5,350,689 each incorporated herein by

reference) or microprojectile bombardment (e. g., U. S. Patent 5,736,369; U. S. Patent 5,610,042; U. S. Patent 5,554,798; U. S. Patent 5,550,318; U. S. Patent 5,015,580; U. S. Patent 5,324,646; U. S. Patent 5,484,956; U. S. Patent 5,489,520; U. S. Patent 5,538,880, each incorporated herein by reference). Transformation techniques have been widely described in art, for example, U. S. Patent 5,792,935 (incorporated herein by reference) describes the transformation of plants using Agrobacterium tumefaciens. Additional disclosure regarding promoters and plant cell transformation may be found in U. S. Patent 5,780,709; U. S. Patent 5,780,708; U. S. Patent 5,767,376; U. S. Patent 5,750,870; U. S. Patent 5,736,369, U. S. Patent 5,405,765; U. S. Patent 5,508,468; U. S. Patent 5,538,877, each incorporated herein by reference. The expression of the polypeptide is directed specifically to the seeds of the transgenic plant.

A transgenic plant of the present invention produced from a plant cell transformed with a tissue specific promoter can be crossed with a second transgenic plant developed from a plant cell transformed with a different tissue specific promoter to produce a hybrid transgenic plant that shows the effects of transformation in more than one specific tissue.

Exemplary tissue-specific promoters are corn sucrose synthetase 1 (Yang et al., 1990), corn alcohol dehydrogenase 1 (Vogel et al., 1989), corn light harvesting complex (Simpson, 1986), corn heat shock protein (Odell et al., 1985), pea small subunit RuBP carboxylase (Poulsen et al., 1986; Cashmore et al., 1983), acetyl CoA carboxylase promoter (U. S. Patent 5,539,092), Ti plasmid mannopine synthase (Langridge et al., 1989), Ti plasmid nopaline synthase (Langridge et al., 1989), petunia chalcone isomerase (Van Tunen et al., 1988), bean glycine rich protein 1, CaMV 35s transcript (Odell et al., 1985) and Potato patatin (Wenzler et al., 1989), root specific promoters are disclosed in U. S. Patent 5,792,925, incorporated herein by reference. Preferred promoters are the cauliflower mosaic virus (CaMV 35S) promoter, a pollen specific promoter (U. S. Patent 5,545,546,5,412,085 incorporated herein by reference) and the rice actin promoter.

The choice of which expression vector and ultimately to which promoter a polypeptide coding region is operatively linked depends directly on the functional properties desired, e. g., the location and timing of protein expression, and the host cell to be transformed. These are well known limitations inherent in the art of constructing recombinant DNA molecules. However, a vector useful in practicing the present invention is capable of directing the expression of the polypeptide coding region to which it is operatively linked.

Typical vectors useful for expression of genes in higher plants are well known in the art and include vectors derived from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens described (Rogers et al., 1987). However, several other plant integrating vector systems are known to function in plants including pCaMVCN transfer control vector described (Fromm et nul.. 1985). Plasmid pCaMVCN (available from Pharmacia, Piscataway, NJ) includes the cauliflower mosaic virus CaMV 35S promoter.

In preferred embodiments, the vector used to express the polypeptide includes a selection marker that is effective in a plant cell, preferably a drug resistance selection marker. One preferred drug resistance marker is the gene whose expression results in kanamycin resistance; i. e., the chimeric gene containing the nopaline synthase promoter, Tn5 neomycin phosphotransferase II (nptlI) and nopaline synthase 3'non-translated region described (Rogers et al., 1988). Another preferred selection marker is the bar gene, which confers glyphosphate esistance. Selectable markers useful in plant transformation are disclosed in U. S. Patent 5,741,665; U. S. Patent 5,719,046; U. S. Patent 5,661,017; U. S. Patent U. S. Patent 5,633,444; U. S. Patent U. S. Patent 5,608,147 ; U. S. Patent 5.597,717; U. S. Patent 5,595,896; U. S. Patent 5,589,611; U. S. Patent 5,527,674; U. S. Patent U. S. Patent 5,470,729; U. S. Patent 5,432,079; U. S. Patent U. S. Patent 5,217,902; U. S. Patent 5,073,675; U. S. Patent 4,784,949; U. S. Patent 4,771,002 (each specifically incorporated herein by reference).

RNA polymerase transcribes a coding DNA sequence through a site where polyadenylation occurs. Typically, DNA sequences located a few hundred base pairs downstream of the polyadenylation site serve to terminate transcription. Those DNA sequences are referred to herein as transcription-termination regions. Those regions are required for efficient polyadenylation of transcribed messenger RNA (mRNA).

Means for preparing expression vectors are well known in the art. Expression (transformation vectors) used to transform plants and methods of making those vectors are described in U. S. Patent 4,971,908, U. S. Patent 4,940,835, U. S. Patent 4,769,061 and U. S. Patent 4,757,011, the disclosures of which are incorporated herein by reference. Those vectors can be modified to include a coding sequence in accordance with the present invention.

A variety of methods have been developed to operatively link DNA to vectors via complementary cohesive termini or blunt ends. For instance, complementary homopolymer tracts can be added to the DNA segment to be inserted and to the vector DNA. The vector and DNA segment are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.

A coding region that encodes a polypeptide having the ability to confer increased levels of lignin biosynthesis to a cell is preferably a tomato BEAT gene. In preferred embodiments, such a polypeptide has the amino acid residue sequence of SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or a functional equivalent of that sequence.

In other embodiments a BEAT gene of the current invention may be operable linked to a transit peptide. In a particular embodiment, this transit peptide may be directed to the chloroplast. The transit peptide may, for example, be a heterologous transit peptide such as that of the small subunit of ribulose bisphosphate carboxylase.

In still other embodiments of the current invention, a transit peptide operable linked to a BEAT gene may be directed to locations within a plant cell other than the flowers.

VIII. Transgenic BEAT Plants and Plant Cells The present invention provides methods for producing a transgenic plant which comprises a nucleic acid segment encoding at least a portion of the BEAT gene of the present invention. The process of producing transgenic plants is well-known in the art. In general, the method comprises transforming a suitable host cell with a DNA segment which contains a promoter operatively linked to a coding region that encodes a BEAT gene. Such a coding region is generally operatively linked to a transcription-terminating region, whereby the promoter is capable of driving the transcription of the coding region in the cell, and hence providing the cell the ability to produce the recombinant protein in vivo. Alternatively, in instances where it is desirable to control, regulate, or decrease the amount of a particular recombinant protein expressed in a particular transgenic cell, the invention also provides for the expression of BEAT protein antisense mRNA. The use of antisense mRNA as a means of controlling or decreasing the amount of a given protein of interest in a cell is well-known in the art.

Another aspect of the invention comprises transgenic plants which express a gene or gene segment encoding one or more of the novel polypeptide compositions disclosed herein. As used herein, the term"transgenic plant"is intended to refer to a plant that has incorporated DNA sequences, which are in addition to those originally present, DNA sequences not normally transcribed into RNA or translated into a protein ("expressed"), or any other genes or DNA sequences which one desires to introduce into the non-transformed plant, such as genes which may normally be present in the non-transformed plant but which one desires to either genetically engineer or to have altered expression.

It is contemplated that in some instances the genome of a transgenic plant of the present invention will have been augmented through the stable introduction of one or more lignin biosynthesis genes, either native, synthetically modified, or mutated. In some instances, more than one transgene will be incorporated into the genome of the

transformed host plant cell. Such is the case when more than one lignin biosynthesis protein-encoding DNA segment is incorporated into the genome of such a plant. In certain situations, it may be desirable to have one, two, three, four, five or even more lignin biosynthesis proteins (either native or recombinantly-engineered) incorporated and stably expressed in the transformed transgenic plant.

A preferred gene which may be introduced includes, for example, a BEAT gene in order to reduce the clove-like taste and flavor in plants. Still other preferred genes are those which, when introduced into a plant or plant cell, result in increased acetylation of benzylalcohol, which will alter the flavor of the plant. These genes may be derived from wild-type BEAT, or may be chimeras of BEAT and other enzymes in the flavor producing pathways.

Means for transforming a plant cell and the preparation of a transgenic cell line are well-known in the art, and are discussed herein. Vectors, plasmids, cosmids, YACs (yeast artificial chromosomes) and DNA segments for use in transforming such cells will, of course, generally comprise either the operons, genes, or gene-derived sequences of the present invention, either native, or synthetically-derived, and particularly those encoding the disclosed crystal proteins. These DNA constructs can further include structures such as promoters, enhancers, polylinkers, or even gene sequences which have positively-or negatively-regulating activity upon the particular genes of interest as desired. The DNA segment or gene may encode either a native or modified crystal protein, which will be expressed in the resultant recombinant cells, and/or which will impart an improved phenotype to the regenerated plant.

Such transgenic plants may be desirable for their decreased clove-like flavor and fragrance, resulting from the incorporation of one or more DNA segments encoding one or more BEAT and other phenylpropanoid biosynthesis genes.

Particularly preferred plants include tomato and other plants the like.

In a related aspect, the present invention also encompasses a seed produced by the transformed plant, a progeny from such seed, and seed produced by the progeny of the original transgenic plant, produced in accordance with the above process. Such progeny and seeds will have a lignin or phenylpropanoid biosynthesis protein- encoding transgene stably incorporated into their genome, and such progeny plants will inherit the traits afforded by the introduction of a stable transgene in Mendelian fashion. All such transgenic plants having incorporated into their genome transgenic DNA segments encoding one or more lignin or phenylpropanoid biosynthesis proteins or polypeptides are aspects of this invention.

A. Sources of Cells Practicing the present invention includes the generation and use of recipient cells. As used herein, the term"recipient cells"refers to cells that are receptive to transformation and subsequent regeneration into stably transformed, fertile plants.

Plant recipient cell targets include, but are not limited to, meristem cells, Type I, Type II, and Type III callus, immature embryos and gametic cells such as microspores pollen, sperm and egg cells. Type I, Type II, and Type III callus may be initiated from tissue sources including, but not limited to, immature embryos, seedling apical meristems, microspores and the such. Those cells which are capable of proliferating as callus are also recipient cells for genetic transformation. Pollen, as well as its precursor cells, microspores, may be capable of functioning as recipient cells for genetic transformation, or as vectors to carry foreign DNA for incorporation during fertilization. Direct pollen transformation would obviate the need for cell culture.

Meristematic cells (i. e., plant cells capable of continual cell division and characterized by an undifferentiated cytological appearance, normally found at growing points or tissues in plants such as root tips, stem apices, lateral buds, etc.) may represent another type of recipient plant cell. Because of their undifferentiated growth and capacity for organ differentiation and totipotency, a single transformed meristematic cell could be recovered as a whole transformed plant. In fact, it is proposed that embryogenic suspension cultures may be an in vitro meristematic cell system,

retaining an ability for continued cell division in an undifferentiated state, controlled by the media environment.

In certain embodiments, cultured plant cells that can serve as recipient cells for transforming with desired DNA segments include corn cells, and cells from Zea mays L. Somatic cells are of various types. Embryogenic cells are one example of somatic cells which may be induced to regenerate a plant through embryo formation. Non- embryogenic cells are those which will typically not respond in such a fashion. An example of non-embryogenic cells are certain Black Mexican Sweet (BMS) corn cells.

The development of embryogenic calli and suspension cultures useful in the context of the present invention, e. g., as recipient cells for transformation, has been described in U. S. Patent 5,134,074, incorporated herein by reference.

The present invention also provides certain techniques that may enrich recipient cells within a cell population. For example, Type II callus development, followed by manual selection and culture of friable, embryogenic tissue, generally results in an enrichment of recipient cells for use in, e. g., micro-projectile transformation. Suspension culturing, particularly using the media disclosed herein, may also improve the ratio of recipient to non-recipient cells in any given population.

Manual selection techniques which are employed to select recipient cells may include, e. g., assessing cell morphology and differentiation, or may use various physical or biological means. Cryopreservation is also contemplated as a possible method of selecting for recipient cells.

Manual selection of recipient cells, e. g., by selecting embryogenic cells from the surface of a Type II callus, is one means employed by the inventors in an attempt to enrich for recipient cells prior to culturing (whether cultured on solid media or in suspension). The preferred cells may be those located at the surface of a cell cluster, and may further be identifiable by their lack of differentiation, their size and dense

cytoplasm. The preferred cells will generally be those cells which are less differentiated, or not yet committed to differentiation. Thus, one may wish to identify and select those cells which are cytoplasmically dense, relatively unvacuolated with a high nucleus to cytoplasm ratio (e. g., determined by cytological observations), small in size (e. g., 10-20 mm), and capable of sustained divisions and somatic proembryo formation.

It is proposed that other means for identifying such cells may also be employed. For example, through the use of dyes, such as Evan's blue, which are excluded by cells with relatively non-permeable membranes, such as embryogenic cells, and taken up by relatively differentiated cells such as root-like cells and snake cells (so-called due to their snake-like appearance).

Other possible means of identifying recipient cells include the use of isozyme markers of embryogenic cells, such as glutamate dehydrogenase, which can be detected by cytochemical stains (Fransz et al., 1989). However, it is cautioned that the use of isozyme markers such as glutamate dehydrogenase may lead to some degree of false positives from non-embryogenic cells such as root cells which nonetheless have a relatively high metabolic activity.

B. Media In certain embodiments, recipient cells are selected following growth in culture. Where employed, cultured cells will preferably be grown either on solid supports or in the form of liquid suspensions. In either instance, nutrients may be provided to the cells in the form of media, and environmental conditions controlled.

There are many types of tissue culture media comprised of amino acids, salts, sugars, growth regulators and vitamins. Most of the media employed in the practice of the invention will have some similar components, the media differ in the composition and proportions of their ingredients depending on the particular application envisioned. For example, various cell types usually grow in more than one type of media, but will

exhibit different growth rates and different morphologies, depending on the growth media. In some media, cells survive but do not divide.

Various types of media suitable for culture of plant cells have been previously described. Examples of these media include, but are not limited to, the N6 medium described by Chu et al. (1975) and MS media (Murashige and Skoog, 1962). Media such as MS which have a high ammonia/nitrate ratio are counterproductive to the generation of recipient cells in that they promote loss of morphogenic capacity. N6 media, on the other hand, has a somewhat lower ammonia/nitrate ratio, and is contemplated to promote the generation of recipient cells by maintaining cells in a proembryonic state capable of sustained divisions.

C. Cell Cultures 1. Initiation In the practice of the invention it is sometimes, but not always, necessary to develop cultures which contain recipient cells. Suitable cultures can be initiated from a number of whole plant tissue explants including, but not limited to, immature embryos, leaf bases, immature tassels, anthers, microspores, and other tissues containing cells capable of in vitro proliferation and regeneration of fertile plants. For example, recipient cell cultures are initiated from immature embryos by growing excised immature embryos on a solid culture medium containing growth regulators including, but not limited to, dicamba., 2,4-D, NAA, and IAA. In some instances it will be preferred to add silver nitrate to culture medium for callus initiation as this compound has been reported to enhance culture initiation (Vain et al., 1989).

Embryos will produce callus that varies greatly in morphology including from highly unorganized cultures containing very early embryogenic structures, to highly organized cultures containing large late embryogenic structures. This variation in culture morphology may be related to genotype, culture medium composition, size of the initial embryos and other factors. Each of these types of culture morphologies is a source of recipient cells.

The development of suspension cultures capable of plant regeneration may be used in the context of the present invention. Suspension cultures may be initiated by transferring callus tissue to liquid culture medium containing growth regulators.

Addition of coconut water or other substances to suspension culture medium may enhance growth and culture morphology, but the utility of suspension cultures is not limited to those containing these compounds. In some embodiments of this invention, the use of suspension cultures will be preferred as these cultures grow more rapidly and are more easily manipulated than callus cells growing on solid culture medium.

When immature embryos or other tissues directly removed from a whole plant are used as the target tissue for DNA delivery, it will only be necessary to initiate cultures of cells insofar as is necessary for identification and isolation of transformants. In an illustrative embodiment, DNA is introduced by particle bombardment into immature embryos following their excision from the plant.

Embryos are transferred to a culture medium that will support proliferation of tissues and allow for selection of transformed sectors, 0-14 days following DNA delivery. In this embodiment of the invention it is not necessary to establish stable callus cultures capable of long term maintenance and plant regeneration.

2. Maintenance The method of maintenance of cell cultures may contribute to their utility as sources of recipient cells for transformation. Manual selection of cells for transfer to fresh culture medium, frequency of transfer to fresh culture medium, composition of culture medium, and environment factors including, but not limited to, light quality and quantity and temperature are all important factors in maintaining callus and/or suspension cultures that are useful as sources of recipient cells. It is contemplated that alternating callus between different culture conditions may be beneficial in enriching for recipient cells within a culture. For example, it is proposed that cells may be cultured in suspension culture, but transferred to solid medium at regular intervals.

After a period of growth on solid medium cells can be manually selected for return to liquid culture medium. It is proposed that by repeating this sequence of transfers to

fresh culture medium it is possible to enrich for recipient cells. It is also contemplated that passing cell cultures through a 1.9 mm sieve is useful in maintaining the friability of a callus or suspension culture and may be beneficial is enriching for transformable cells.

3. Cryopreservation Additionally, the inventors propose that cryopreservation may effect the development of, or perhaps select for, recipient cells. Cryopreservation selection may operate due to a selection against highly vacuolated, non-embryogenic cells, which may be selectively killed during cryopreservation. The inventors propose that there is a temporal window in which cultured cells retain their regenerative ability, thus, it is believed that they must be preserved at or before that temporal period if they are to be used for future transformation and regeneration.

For use in transformation, suspension or callus culture cells may be cryopreserved and stored for periods of time, thawed, then used as recipient cells for transformation. An illustrative embodiment of cryopreservation methods comprises the steps of slowly adding cryoprotectants to suspension cultures to give a final concentration of 10% dimethyl sulfoxide, 10% polyethylene glycol (6000MW), 0.23 M proline and 0.23 M glucose. The mixture is then cooled to-35°C at 0.5°C per min. After an isothermal period of 45 min, samples are placed in liquid N2 (modification of methods of Withers and King (1979); and Finkle et al. (1985)). To reinitiate suspension cultures from cryopreserved material, cells may be thawed rapidly and pipetted onto feeder plates similar to those described by Rhodes et al. (Vaeck et al., 1987).

IX. DNA Sequences Virtually any DNA composition may be used for delivery to recipient cells by modem transformation techniques to ultimately produce fertile transgenic plants. In accordance with the present invention the genes used to create transgenic plants will be BEAT genes of phenylpropanoid biosynthesis. By way of example, DNA

segments in the form of vectors and plasmids, or linear DNA fragments, in some instances containing only the BEAT gene DNA element to be expressed in the plant, and the like, may be employed.

In certain embodiments, it is contemplated that one may wish to employ replication-competent viral vectors in monocot transformation. Such vectors include, for example, wheat dwarf virus (WDV)"shuttle"vectors, such as pWl-11 and PW1- GUS (Ugaki et al., 1991). These vectors are capable of autonomous replication in plant cells as well as E. coli, and as such may provide increased sensitivity for detecting DNA delivered to transgenic cells. A replicating vector may also be useful for delivery of genes flanked by DNA sequences from transposable elements such as Ac, Ds, or Mu. It has been proposed (Laufs eí al., 1990) that transposition of these elements within the plant genome requires DNA replication. It is also contemplated that transposable elements would be useful for introducing DNA fragments lacking elements necessary for selection and maintenance of the plasmid vector in bacteria, e. g., antibiotic resistance genes and origins of DNA replication. It is also proposed that use of a transposable element such as Ac, Ds, or Mu would actively promote integration of the desired DNA and hence increase the frequency of stably transformed cells.

Vectors, plasmids, cosmids, YACs (yeast artificial chromosomes) and DNA segments for use in transforming such cells will, of course, generally comprise the cDNA, gene or genes which one desires to introduce into the cells. These DNA constructs can further include structures such as promoters, enhancers, polylinkers, or even regulatory genes as desired. The DNA segment or gene chosen for cellular introduction will encode a protein involved in phenylpropanoid biosynthesis and will be expressed _in the resultant recombinant cells, such as will result in a screenable or selectable trait and/or which will impart an improved phenotype to the regenerated plant.

A. Regulatory Elements The construction of vectors which may be employed in conjunction with the present invention will be known to those of skill of the art in light of the present disclosure (see e. g., Sambrook et al., 1989; Gelvin et al., 1990). Preferred constructs will generally include a plant promoter such as the CaMV 3 5 S promoter (Odell et al., 1985), or others such as CaMV 19S (Lawton et al., 1987), nos (Ebert et al., 1987), Adh (Walker et al., 1987), sucrose synthase (Yang and Russell, 1990), a-tubulin, actin (Wang et al., 1992), cab (Sullivan et al., 1989), PEPCase (Hudspeth and Grula, 1989) or those associated with the R gene complex (Chandler et al., 1989). Tissue specific promoters such as root cell promoters (Conkling et al., 1990) and tissue specific enhancers (Fromm et al., 1989) are also contemplated to be particularly useful, as are inducible promoters such as ABA-and turgor-inducible prompters.

Constructs will also include the gene of interest along with a 3'end DNA sequence that acts as a signal to terminate transcription and allow for the poly- adenylation of the resultant mRNA. The most preferred 3'elements are contemplated to be those from the nopaline synthase gene of Agrobacterium tumefasciens (Bevan et al., 1983), the terminator for the T7 transcript from the octopine synthase gene of Agrobacterium tumefasciens, and the 3'end of the protease inhibitor I or 11 genes from potato or tomato. Regulatory elements such as Adh intron 1 (Callis et al., 1987), sucrose synthase intron (Vasil et al., 1989) or TMV omega element (Gallie, et al., 1989), or actin intron (Wang et al., 1992), may further be included where desired.

As the DNA sequence between the transcription initiation site and the start of the coding sequence, i. e., the untranslated leader sequence, can influence gene expression, one may also wish to employ a particular leader sequence. Preferred leader sequences are contemplated to include those which include sequences predicted to direct optimum expression of the attached gene, i. e., to include a preferred consensus leader sequence which may increase or maintain mRNA stability and prevent inappropriate initiation of translation. The choice of such sequences will be

known to those of skill in the art in light of the present disclosure. Sequences that are derived from genes that are highly expressed in plants will be most preferred.

It is contemplated that vectors for use in accordance with the present invention may be constructed to include the ocs enhancer element. This element was first identified as a 16 bp palindromic enhancer from the octopine synthase (ocs) gene of agrobacterium (Ellis et al., 1987), and is present in at least 10 other promoters (Bouchez et al., 1989). It is proposed that the use of an enhancer element, such as the ocs element and particularly multiple copies of the element, will act to increase the level of transcription from adjacent promoters when applied in the context of monocot transformation.

It is specifically envisioned that BEAT genes may be introduced under the control of novel promoters or enhancers, etc., or perhaps even homologous or tissue specific (e. g., root-, collar/sheath-, whorl-, stalk-, earshank-, kernel-or leaf-specific) promoters or control elements. Indeed, it is envisioned that a particular use of the present invention will be the targeting of lignin or phenylpropanoid biosynthesis in a tissue-specific manner. For example, insect resistant genes may be expressed specifically in the whorl and collar/sheath tissues which are targets for the first and second broods, respectively, of ECB.

Vectors for use in tissue-specific targeting of genes in transgenic plants will typically include tissue-specific promoters and may also include other tissue-specific control elements such as enhancer sequences. Promoters which direct specific or enhanced expression in certain plant tissues will be known to those of skill in the art in light of the present disclosure. These include, for example, the rbcS promoter, specific for green tissue; the ocs, nos and mas promoters which have higher activity in roots or wounded leaf tissue; a truncated (-90 to +8) 35S promoter which directs enhanced expression in roots, an a-tubulin gene that directs expression in roots and promoters derived from zein storage protein genes which direct expression in endosperm. It is particularly contemplated that one may advantageously use the 16 bp

ocs enhancer element from the octopine synthase (ocs) gene (Ellis et al., 1987; Bonchez et al., 1989), especially when present in multiple copies, to achieve enhanced expression in roots.

It is also contemplated that tissue specific expression may be functionally accomplished by introducing a constitutively expressed gene (all tissues) in combination with an antisense gene that is expressed only in those tissues where the gene product is not desired. For example, a gene coding for a BEAT gene may be introduced such that it is expressed in all tissues using the 35S promoter from Cauliflower Mosaic Virus.

Alternatively, one may wish to obtain novel tissue-specific promoter sequences for use in accordance with the present invention. To achieve this, one may first isolate cDNA clones from the tissue concerned and identify those clones which are expressed specifically in that tissue, for example, using Northern blotting. Ideally, one would like to identify a gene that is not present in a high copy number, but which gene product is relatively abundant in specific tissues. The promoter and control elements of corresponding genomic clones may then be localized using the techniques of molecular biology known to those of skill in the art.

It is contemplated that expression of BEAT genes in transgenic plants may in some cases be desired only under specified conditions. It is contemplated that expression of such genes at high levels may have detrimental effects. It is known that a large number of genes exist that respond to the environment. For example, expression of some genes such as rbcS, encoding the small subunit of ribulose bisphosphate carboxylase, is regulated by light as mediated through phytochrome.

Other genes are induced by secondary stimuli. A number of genes have been shown to be induced by ABA (Skriver and Mundy, 1990). It is also expected, in particular embodiments, that inducible expression of BEAT genes in transgenic plants may be desired.

It is proposed that in some embodiments of the present invention expression of a BEAT gene in a transgenic plant will be desired only in a certain time period during the development of the plant. Developmental timing is frequently correlated with tissue specific gene expression. For example, expression of zein storage proteins is initiated in the endosperm about 15 days after pollination.

Additionally, vectors may be constructed and employed in the intracellular targeting of a specific gene product within the cells of a transgenic plant or in directing a protein to the extracellular environment. This will generally be achieved by joining a DNA sequence encoding a transit or signal peptide sequence to the coding sequence of a particular gene. The resultant transit, or signal, peptide will transport the protein to a particular intracellular, or extracellular destination, respectively, and will then be post-translationally removed. Transit or signal peptides act by facilitating the transport of proteins through intracellular membranes, e. g., vacuole, vesicle, plastid and mitochondrial membranes, whereas signal peptides direct proteins through the extracellular membrane.

A particular example of such a use concerns the direction of a herbicide resistance gene, such as the EPSPS gene, to a particular organelle such as the chloroplast rather than to the cytoplasm. This is exemplified by the use of the rbcS transit peptide which confers plastid-specific targeting of proteins. In addition, it is proposed that it may be desirable to target BEAT genes to the extracellular spaces or to the vacuole.

It is also contemplated that it may be useful to target DNA itself within a cell.

For example, it may be useful to target introduced DNA to the nucleus as this may increase the frequency of transformation. Within the nucleus itself it would be useful to target a gene in order to achieve site specific integration. For example, it would be useful to have an gene introduced through transformation replace an existing gene in the cell.

B. Marker Genes In order to improve the ability to identify transformants, one may desire to employ a selectable or screenable marker gene as, or in addition to, the expressible gene of interest."Marker genes"are genes that impart a distinct phenotype to cells expressing the marker gene and thus allow such transformed cells to be distinguished from cells that do not have the marker. Such genes may encode either a selectable or screenable marker, depending on whether the marker confers a trait which one can select for by chemical means, i. e., through the use of a selective agent (e. g., a herbicide, antibiotic, or the like), or whether it is simply a trait that one can identify through observation or testing, i. e., by screening (e. g., the R-locus trait). Of course, many examples of suitable marker genes are known to the art and can be employed in the practice of the invention.

Included within the terms selectable or screenable marker genes are also genes which encode a"secretable marker"whose secretion can be detected as a means of identifying or selecting for transformed cells. Examples include markers which encode a secretable antigen that can be identified by antibody interaction, or even secretable enzymes which can be detected by their catalytic activity. Secretable proteins fall into a number of classes, including small, diffusible proteins detectable, e. g., by ELISA; small active enzymes detectable in extracellular solution (e. g., a-amylase, p-lactamase, phosphinothricin acetyltransferase); and proteins that are inserted or trapped in the cell wall (e. g., proteins that include a leader sequence such as that found in the expression unit of extensin or tobacco PR-S).

With regard to selectable secretable markers, the use of a gene that encodes a protein that becomes sequestered in the cell wall, and which protein includes a unique epitope is considered to be particularly advantageous. Such a secreted antigen marker would ideally employ an epitope sequence that would provide low background in plant tissue, a promoter-leader sequence that would impart efficient expression and targeting across the plasma membrane, and would produce protein that is bound in the

cell wall and yet accessible to antibodies. A normally secreted wall protein modified to include a unique epitope would satisfy all such requirements.

One example of a protein suitable for modification in this manner is extensin, or hydroxyproline rich glycoprotein (HPRG). The use of HPRG (Steifel et al., 1990) which is preferred as this molecule is well characterized in terms of molecular biology, expression and protein structure. However, any one of a variety of extensions and/or glycine-rich wall proteins (Keller et al., 1989) could be modified by the addition of an antigenic site to create a screenable marker.

One exemplary embodiment of a secretable screenable marker concerns the use of a genomic clone encoding a protein of choice, modified to include a unique 15 residue epitope MATVPELNCEMPPSD (SEQ ID NO: 3) from the pro-region of murine interleukin-l-B (IL-1-13). However, virtually any detectable epitope may be employed in such embodiments, as selected from the extremely wide variety of antigen: antibody combinations known to those of skill in the art. The unique extracellular epitope, whether derived from IL-1-13 or any other protein or epitopic substance, can then be straightforwardly detected using antibody labeling in conjunction with chromogenic or fluorescent adjuncts.

Elements of the present disclosure are exemplified in detail through the use of the bar and/or GUS genes, and also through the use of various other markers. Of course, in light of this disclosure, numerous other possible selectable and/or screenable marker genes will be apparent to those of skill in the art in addition to the one set forth hereinbelow. Therefore, it will be understood that the following discussion is exemplary rather than exhaustive. In light of the techniques disclosed herein and the general recombinant techniques which are known in the art, the present invention renders possible the introduction of any gene, including marker genes, into a recipient cell to generate a transformed plant.

C. Selectable Markers Possible selectable markers for use in connection with the present invention include, but are not limited to, a neo gene (Potrykus et al., 1985) which codes for kanamycin resistance and can be selected for using kanamycin, G418, etc.; a bar gene which codes for bialaphos resistance; a mutant gene which encodes an altered EPSP synthase protein (Hinchee et al., 1988) thus conferring glyphosate resistance; a nitrilase gene such as bxn from Klebsiella ozaenae which confers resistance to bromoxynil (Stalker et al., 1988); a mutant acetolactate synthase gene (ALS) which confers resistance to imidazolinone, sulfonylurea or other ALS inhibiting chemicals (European Patent Application 154,204,1985); a methotrexate resistant DHFR gene (Thillet et al., 1988), or a dalapon dehalogenase gene that confers resistance to the herbicide dalapon; or a mutated anthranilate synthase gene that confers resistance to 5-methyl tryptophan. Where a mutant EPSP synthase gene is employed, additional benefit may be realized through the incorporation of a suitable chloroplast transit peptide, CTP (European Pat. No. 0189707).

An illustrative embodiment of a selectable marker gene capable of being used in systems to select transformants is the genes that encode the enzyme phosphinothricin acetyltransferase, such as the bar gene from Streptomyces hygroscopicus or the pat gene from Streptomyces viridochromogenes. The enzyme phosphinothricin acetyl transferase (PAT) inactivates the active ingredient in the herbicide bialaphos, phosphinothricin (PPT). PPT inhibits glutamine synthetase, (Murakami et al., 1986; Twell et al., 1989) causing rapid accumulation of ammonia and cell death.

Where one desires to employ a bialaphos resistance gene in the practice of the invention, the inventors have discovered that a particularly useful gene for this purpose is the bar or pat genes obtainable from species of Streptomyces (e. g., ATCC No.

21,705). The cloning of the bar gene has been described (Murakami et al., 1986; Thompson et al., 1987) as has the use of the bar gene in the context of plants other than monocots (De Block et al., 1987; De Block et al., 1989).

D. Screenable Markers Screenable markers that may be employed include a P-glucuronidase or uidA gene (GUS) which encodes an enzyme for which various chromogenic substrates are known; an R-locus gene, which encodes a product that regulates the production of anthocyanin pigments (red color) in plant tissues (Dellaporta et al., 1988); a p-lactamase gene (Sutcliffe, 1978), which encodes an enzyme for which various chromogenic substrates are known (e. g., PADAC, a chromogenic cephalosporin); a xylE gene (Zukowsky et al., 1983) which encodes a catechol dioxygenase that can convert chromogenic catechols; an oc-amylase gene (Ikuta et al., 1990); a tyrosinase gene (Katz et al., 1983) which encodes an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone which in turn condenses to form the easily-detectable compound melanin; a p-galactosidase gene, which encodes an enzyme for which there are chromogenic substrates; a luciferase (lux) gene (Ow et al., 1986), which allows for bioluminescence detection; a green fluorescent protein (GFP) gene, or even an aequorin gene (Prasher et al., 1985), which may be employed in calcium-sensitivebioluminescencedetection.

A screenable marker contemplated for use in the present invention is firefly luciferase, encoded by the lux gene. The presence of the lux gene in transformed cells may be detected using, for example, X-ray film, scintillation counting, fluorescent spectrophotometry, low-light video cameras, photon counting cameras or multiwell luminometry. It is also envisioned that this system may be developed for populational screening for bioluminescence, such as on tissue culture plates, or even for whole plant screening.

It is further contemplated that a gene encoding green fluorescent protein (GFP) could also be-used as a screenable marker. Cells expressing GFP fluoresce when illuminated with light of particular wavelengths, especially ultraviolet light. Cells or plants expressing GFP can thereby be readily identified..

E. Negative Selectable Markers It is contemplated that in particular embodiments a negative selectable marker may be used with the current invention. It is contemplated that when two or more genes are introduced together by co-transformation that the genes will be linked together on the host chromosome. For example, a gene encoding a BEAT protein that confers a clove-like flavor on the plant may be introduced into a plant together with a bar gene that is useful as a selectable marker and confers resistance to the herbicide Lignite on the plant. However, it may not be desirable to have an insect resistant plant that is also resistant to the herbicide Lignite0. It is proposed that one could also introduce an antisense bar gene that is expressed in those tissues where one does not want expression of the bar gene, e. g., in whole plant parts. Hence, although the bar gene is expressed and is useful as a selectable marker, it is not useful to confer herbicide resistance on the whole plant. The bar antisense gene is a negative selectable marker.

It is also contemplated that a negative selection is necessary in order to screen a population of transformants for rare homologous recombinants generated through gene targeting. For example, a homologous recombinant may be identified through the inactivation of a gene that was previously expressed in that cell. The antisense gene to neomycin phosphotransferase II (nptII) has been investigated as a negative selectable marker in tobacco (Nicotiana tabacum) and Arabidopsis thaliana (Xiang, C. and Guerra, D. J. 1993). In this example both sense and antisense npt II genes are introduced into a plant through transformation and the resultant plants are sensitive to the antibiotic kanamycin. An introduced gene that integrates into the host cell chromosome at the site of the antisense nptII gene, and inactivates the antisense gene, will make the plant resistant to kanamycin and other aminoglycoside antibiotics.

Therefore, rare site specific recombinants may be identified by screening for antibiotic resistance. Similarly, any gene, native to the plant or introduced through transformation, that when inactivated confers resistance to a compound, may be useful as a negative selectable marker.

X. Transformation Techniques A. Electroporation The application of brief, high-voltage electric pulses to a variety of bacterial, animal and plant cells leads to the formation of nanometer-sized pores in the plasma membrane. DNA is taken directly into the cell cytoplasm either through these pores or as a consequence of the redistribution of membrane components that accompanies closure of the pores. Electroporation can be extremely efficient and can be used both for transient expression of cloned genes and for establishment of cell lines that carry integrated copies of the gene of interest. Electroporation, in contrast to calcium phosphate-mediated transfection and protoplast fusion, frequently gives rise to cell lines that carry one, or at most a few, integrated copies of the foreign DNA.

The introduction of DNA by means of electroporation, is well-known to those of skill in the art (U. S. Patent 5,824,302; U. S. Patent 5,629,183; U. S. Patent 5,384,253; U. S. Patent 5,641,664; U. S. Patent 5,712,135 each incorporated herein by reference describe various techniques of electroporation for transforming plants). In this method, certain cell wall-degrading enzymes, such as pectin-degrading enzymes, are employed to render the target recipient cells more susceptible to transformation by electroporation than untreated cells. Alternatively, recipient cells are made more susceptible to transformation, by mechanical wounding. To effect transformation by electroporation one may employ either friable tissues such as a suspension culture of cells, or embryogenic callus, or alternatively, one may transform immature embryos or other organized tissues directly. One would partially degrade the cell walls of the chosen cells by exposing them to pectin-degrading enzymes (pectolyases) or mechanically wounding in a controlled manner. Such cells would then be recipient to DNA transfer by electroporation, which may be carried out at this stage, and transformed cells then identified by a suitable selection or screening protocol dependent on the nature of the newly incorporated DNA.

B. Microprojectile Bombardment A further advantageous method for delivering transforming DNA segments to plant cells is microprojectile bombardment. In this method, particles may be coated with nucleic acids and delivered into cells by a propelling force. Exemplary particles include those comprised of tungsten, gold, platinum and the like.

An advantage of microprojectile bombardment, in addition to it being an effective means of reproducibly stably transforming monocots, is that neither the isolation of protoplasts (Christou et al., 1988) nor the susceptibility to Agrobacterium infection is required. An illustrative embodiment of a method for delivering DNA into maize cells by acceleration is a Biolistics Particle Delivery System, which can be used to propel particles coated with DNA or cells through a screen, such as a stainless steel or Nytex screen, onto a filter surface covered with corn cells cultured in suspension. The screen disperses the particles so that they are not delivered to the recipient cells in large aggregates. It is believed that a screen intervening between the projectile apparatus and the cells to be bombarded reduces the size of projectiles aggregate and may contribute to a higher frequency of transformation by reducing damage inflicted on the recipient cells by projectiles that are too large.

For the bombardment, cells in suspension are preferably concentrated on filters or solid culture medium. Alternatively, immature embryos or other target cells may be arranged on solid culture medium. The cells to be bombarded are positioned at an appropriate distance below the macroprojectile stopping plate. If desired, one or more screens are also positioned between the acceleration device and the cells to be bombarded. Through the use of techniques set forth herein one may obtain up to 1000 or more foci of cells transiently expressing a marker gene. The number of cells in a focus which express the exogenous gene product 48 hours post-bombardment often range from 1 to 10 and average 1 to 3.

In bombardment transformation, one may optimize the prebombardment culturing conditions and the bombardment parameters to yield the maximum numbers

of stable transformants. Both the physical and biological parameters for bombardment are important in this technology. Physical factors are those that involve manipulating the DNA/microprojectile precipitate or those that affect the flight and velocity of either the macro-or microprojectiles. Biological factors include all steps involved in manipulation of cells before and immediately after bombardment, the osmotic adjustment of target cells to help alleviate the trauma associated with bombardment, and also the nature of the transforming DNA, such as linearized DNA or intact supercoiled plasmids. It is believed that pre-bombardment manipulations are especially important for successful transformation of immature embryos.

Accordingly, it is contemplated that one may wish to adjust various of the bombardment parameters in small scale studies to fully optimize the conditions. One may particularly wish to adjust physical parameters such as gap distance, flight distance, tissue distance, and helium pressure. One may also minimize the trauma reduction factors (TRFs) by modifying conditions which influence the physiological state of the recipient cells and which may therefore influence transformation and integration efficiencies. For example, the osmotic state, tissue hydration and the subculture stage or cell cycle of the recipient cells may be adjusted for optimum transformation. The execution of other routine adjustments will be known to those of skill in the art in light of the present disclosure.

C. Agrobacterium-Mediated Transfer Agrobacterium-mediated transfer is a widely applicable system for introducing genes into plant cells because the DNA can be introduced into whole plant tissues, thereby bypassing the need for regeneration of an intact plant from a protoplast. The use of Agrobacterium-mediated plant integrating vectors to introduce DNA into plant cells is well known in the art. See, for example, the methods described (Fraley et al., 1985; Rogers et al., 1987). Further, the integration of the Ti-DNA is a relatively precise process resulting in few rearrangements. The region of DNA to be transferred is defined by the border sequences, and intervening DNA is usually inserted into the plant genome as described (Spielmann et al., 1986; Jorgensen et al., 1987).

Modem Agrobacterium transformation vectors are capable of replication in E. coli as well as Agrobacterium, allowing for convenient manipulations as described in Klee et al., 1985. Moreover, recent technological advances in vectors for Agrobacterium-mediated gene transfer have improved the arrangement of genes and restriction sites in the vectors to facilitate construction of vectors capable of expressing various polypeptide coding genes. The vectors described (Rogers et al., 1987), have convenient multi-linker regions flanked by a promoter and a polyadenylation site for direct expression of inserted polypeptide coding genes and are suitable for present purposes. In addition, Agrobacterium containing both armed and disarmed Ti genes can be used for the transformations. In those plant strains where Agrobacterium-mediated transformation is efficient, it is the method of choice because of the facile and defined nature of the gene transfer.

Agrobacterium-mediated transformation of leaf disks and other tissues such as cotyledons and hypocotyls appears to be limited to plants that Agrobacterium naturally infects. Agrobacterium-mediated transformation is most efficient in dicotyledonous plants. Few monocots appear to be natural hosts for Agrobacterium, although transgenic plants have been produced in asparagus using Agrobacterium vectors as described (Bytebier et al., 1987). Therefore, commercially important cereal grains such as rice, corn, and wheat must usually be transformed using alternative methods. Agrobacterium-mediated transformation of maize has, however, recently been described in U. S. Pat. No. 5,591,616, which is specifically incorporated herein by reference. Further, it has been shown that homozygous BEAT mutants are more susceptible to Agrobacterium-mediated transformation.

A transgenic plant formed using Agrobacterium transformation methods typically contains a single transgene or a few copies of a transgene on one chromosome. Such transgenic plants can be referred to as being heterozygous for the added gene. However, inasmuch as use of the word"heterozygous"usually implies the presence of a complementary gene at the same locus of the second chromosome of

a pair of chromosomes, and there is no such gene in a plant containing one added gene as here, it is believed that a more accurate name for such a plant is hemizygous.

More preferred is a transgenic plant that is homozygous for the added structural gene; i. e., a transgenic plant that contains two added genes, one gene at the same locus on each chromosome of a chromosome pair. A homozygous transgenic plant can be obtained by sexually mating (selfing) a hemizygous transgenic plant that contains a single added gene, germinating some of the seed produced and analyzing the resulting plants produced for enhanced carboxylase activity relative to a control (native, non-transgenic) or a hemizygous transgenic plant.

It is to be understood that two different transgenic plants can also be mated to produce offspring that contain multiple independently segregating added, exogenous genes. Specifically contemplated by the inventors, is the creation of plants which contain 1,2,3,4,5, or even more independently segregating added, exogenous genes.

Selfing of appropriate progeny can produce plants that are homozygous for all added, exogenous genes that encode a polypeptide of interest. Back-crossing to a parental plant and out-crossing with a non-transgenic plant are also contemplated.

D. Other Transformation Methods Transformation of plant protoplasts can be achieved using methods based on calcium phosphate precipitation, polyethylene glycol treatment, electroporation, and combinations of these treatments (see, e. g., Potrykus et al., 1985; Lorz et al., 1985; Fromm et al., 1986; Uchimiya et al., 1986; Callis et al., 1987; Marcotte et al., 1988).

Application of these systems to different plant strains depends upon the ability to regenerate that particular plant strain from protoplasts. Illustrative methods for the regeneration of cereals from protoplasts are described (Fujimura et al., 1985; Toriyama et al., 1986; Yamada et al., 1986; Abdullah et al., 1986).

To transform plant strains that cannot be successfully regenerated from protoplasts, other ways to introduce DNA into intact cells or tissues can be utilized.

For example, regeneration of cereals from immature embryos or explants can be effected as described (Vasil, 1988). In addition,"particle gun"or high-velocity microprojectile technology can be utilized (Vasil, 1992).

Using that latter technology, DNA is carried through the cell wall and into the cytoplasm on the surface of small metal particles as described (Klein et al., 1987; Klein et al., 1988; McCabe et al., 1988). The metal particles penetrate through several layers of cells and thus allow the transformation of cells within tissue explants.

XI. Methods for Producing Plants With Enhanced Fragrance and Flavor By transforming a suitable host cell, such as a plant cell, with a recombinant BEAT gene-containing segment, expression of the encoded BEAT gene (i. e., a protein catalyzing benzylacetate biosynthesis) can result in increased production of benzylacetate, thereby increasing the stone-fruit-like flavor thereof.

It is also contemplated by the inventors that particular combinations of transformed biosynthesis genes, for example, those of the phenylpropanoid biosynthesis, may be created by standard plant breeding methods, which are well known in the art. Such breeding protocols may be aided by the use of genetic markers which are closely linked to the genes of interest.

A preferred method for transformation with BEAT genes envisioned by the inventors is microprojectile bombardment. Techniques for microprojectile bombardment are well known in the art, and are described in Lundquist et al., U. S.

Pat. No. and Adams et al. U. S. Pat. No. 5,489,520, which are specifically incorporated herein by reference.

By way of example, one may utilize an expression vector containing a coding region for a BEAT gene and an appropriate selectable marker to transform a

suspension of embryogenic plant cells, such as wheat or corn cells using a method such as particle bombardment (Maddock et al., 1991; Vasil et al., 1992) to deliver the DNA coated on microprojectiles into the recipient cells. Transgenic plants are then regenerated from transformed embryogenic calli that express the proteins.

The formation of transgenic plants may also be accomplished using other methods of cell transformation which are known in the art such as Agrobacterium- mediated DNA transfer (Fraley et al., 1983). Alternatively, DNA can be introduced into plants by direct DNA transfer into pollen (Zhou et al., 1983; Hess, 1987; Luo et al., 1988), by injection of the DNA into reproductive organs of a plant (Pena et al., 1987), or by direct injection of DNA into the cells of immature embryos followed by the rehydration of desiccated embryos (Neuhaus et al., 1987; Benbrook et al., 1986).

The regeneration, development, and cultivation of plants from single plant protoplast transformants or from various transformed explants is well known in the art (Weissbach et al., 1988). This regeneration and growth process typically includes the steps of selection of transformed cells, culturing those individualized cells through the usual stages of embryonic development through the rooted plantlet stage. Transgenic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are thereafter planted in an appropriate plant growth medium such as soil. Protoplast methods of transformation of maize are described in U. S. Patent 5,350,689.

The development or regeneration of plants containing the foreign, exogenous gene that encodes a polypeptide of interest introduced by Agrobacterium from leaf explants can be achieved by methods well known in the art such as described in Horsch et al., 1985. In this procedure, transformants are cultured in the presence of a selection agent and in a medium that induces the regeneration of shoots in the plant strain being transformed as described (Fraley et al., 1983).

This procedure typically produces shoots within two to four months and those shoots are then transferred to an appropriate root-inducing medium containing the

selective agent and an antibiotic to prevent bacterial growth. Shoots that rooted in the presence of the selective agent to form plantlets are then transplanted to soil or other media to allow the production of roots. These procedures vary depending upon the particular plant strain employed, such variations being well known in the art.

Preferably, the regenerated plants are self-pollinated to provide homozygous transgenic plants, as discussed before. Otherwise, pollen obtained from the regenerated plants is crossed to seed-grown plants of agronomically important, preferably inbred lines. Conversely, pollen from plants of those important lines is used to pollinate regenerated plants. A transgenic plant of the present invention containing a desired polypeptide is cultivated using methods well known to one skilled in the art.

A transgenic plant of this invention thus has an increased amount of the BEAT protein, or the BEAT protein in combination with other flavor/fragrance biosynthesis proteins. A preferred transgenic plant is homozygous and can transmit particular genes and their activities to its progeny. A more preferred transgenic plant is homozygous for the foreign gene or genes, and transmits the gene or genes to all of its offspring on sexual mating. Seed from a transgenic plant may be grown in the field or greenhouse, and resulting sexually mature transgenic plants are self-pollinated to generate true breeding plants. The progeny from these plants become true breeding lines that are evaluated for, by way of example, decreased lignification and increased flavor and fragrance, preferably in the field, under a range of environmental conditions. The inventors contemplate that the present invention will find particular utility in the creation of transgenic plants of commercial interest including various turf grasses, wheat, corn, rice, barley, oats, a variety of ornamental plants and vegetables, as well as a number of nut-and fruit-bearing trees and plants.

XIII. Examples The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the

techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLE 1 Materials and Methods Plant material and growth conditions Details of the construction of true-breeding C. breweri stocks and growing conditions, dynamic headspace collection on Tenax and activated charcoal sorbents, and chemical analyses via GC-MS are as described in Raguso and Pichersky (1995). All headspace collections were performed in a Conviron growth chamber under a 12 hours light/12 hours dark photoperiod. Temperature was set to 22°C during the light period and 18°C during the dark period. In all studies, headspace collections from ambient air and from vegetative tissues were used as controls.

Time Course of Ester Production Volatile production of esters in individual flowers of 4 separate plants was monitored over a 6 day period beginning on the day before anthesis and continuing until floral abscission. Headspace volatiles were collected as described in Raguso and Pichersky (1995). The collections were made at 12 hours intervals, corresponding to the dark and light periods in the growth chamber.

Localization and Quantitation of Emission of Esters in Floral Parts The specific floral parts responsible for scent emission were determined and the emission levels were quantified by headspace collection essentially as described in Raguso and Pichersky (1995). Headspace collection were made from attached, second day (hermaphroditic) intact flowers and from same-stage flowers in which floral organs had been systematically removed, to leave only petals, only anthers or

only the pistil. In order to detect all volatiles emitted by a given flower part, which could possibly emit different compounds at different times, a 24 hours collection period was used.

SAMT and BEAT Enzyme Extraction and Assay Enzyme extraction A crude protein extract was prepared as previously described (Wang et al., 1997). A ratio of 10: 1 (v/w) extraction buffer to tissue fresh weight was used. Protein concentrations in crude extracts were as follow: leaf, 10 mg/mL; sepal, 6.8 mg/mL; petal, 1.4 mg/mL; stigma, 4.1 mg/mL; style, 3.5 mg/mL ; stamen, 13.8 mg/mL. For each time point, flowers from 3 different plats were combined to prepare a crude extract, and at least two independent enzymatic assayed were performed.

SAMT Enzyme Assays and Product Analysis Assay samples were prepared by adding the following to a 1.5-mL microcentrifuge tube: 10 u. L crude extract, 10 iL of 5X assay buffer (250 mM Tris- HC1 [pH 7.5], 14 mM 2-mercaptoethanol), 1 uL of 50 mM salicylic acid, 1.0, ut (2 x 10-5 mCi) of 0.34 mM S- [methyl-4C]-adenosyl-L-methionine (NEN Research Products, Boston, MA), and 28 uL water to bring the assay volume to 50 uL. Assay samples were incubated at 30°C for 30 min in a heating block. The radioactively labeled methylated product was extracted by the addition of 100 uL ethyl acetate, and 20 u. L of the organic phase (on top and clear in color) was transferred to a scintillation vial with 2 ml of non-aqueous scintillation fluid (Bio-Safe NA, Research Products International, Mount Prospect, IL) and counted in a Beckman 2S6800 liquid scintillation counter. The raw data (counts per min) were converted to pkat (picomoles of product produced per sec) based on the specific activity of the substrate and using the-appropriate correction factors for counting efficiency. Controls (for SAMT as well as for BEAT) included assays with boiled crude extracts and with buffers only, and background radioactivity produced in such assays was subtracted from all results. The specificity of SAMT was tested with several related substrates,

such as benzoic acid. No activity was detected with substrates other than salicylic acid.

The identity of the products was verified by several methods. First, 20 u1 of reaction product was spotted on a 10 cm x 20 cm silica gel 60 F254 pre-coated TLC plate (EM Industries, Inc., Gibbstown, NJ), and 5 uL of a 5% (by volume) solution containing authentic standards was spotted on the same plate as a standard. Plates were run and analyzed as previously described (Wang et al., 1997). Products were also analyzed by GC-MS after organic extraction from scaled-up reactions of 1 mL total volume, with both substrates non-radioactive and at a final concentration of 1 mM each. Because esters are hydrolyzed in concentrated acid solutions, the enzymatic assays were not stopped by the addition of concentrated acid as is often done, but HCL hydrolysis (at a final concentration of 0.3 M) was also carried out to distinguish between methylation of the carboxyl OH group vs. methylation of the 2'OH group on the benzyl ring. An enzymatic activity of the latter type was not found in the crude extracts of C. breweri flowers.

BEAT enzyme assays and product analysis Details of the extraction buffers and assay conditions are described herein above. Throughout the purification procedure, enzyme activity was assayed in a total volume of 50 u. l, and the radioactive product was quantified by hexane extraction and scintillation counting as previously described above for SAMT. Product verification was done by radio-TLC and by GC-MS. For the GC-MS analysis, the enzymatic reaction was scaled up to 1 ml final volume of solution containing 50 mM Tris-HCl pH 7.5,3 mM 2-mercaptoethanol, 0.5 mM acetyl CoA (non-radioactive), 1 mM benzylalcohol, and 30 pu ouf purified BEAT from either C. breweri petals or E. coli expression system (approximately 0.5-1.2 mg). Reaction solution was overlaid with 1 ml of hexane, and the reaction was carried out for 1 hour.

Enzyme purification BEAT was purified in a series of chromatographic steps involving the anion exchange column DEAE (DE53, Whatman, Cliffton, NJ), an hydroxyapatite column (Bio-Rad, Hercules, CA), and another anion exchange column, MonoQ, on Pharmacia's FPLC system Pharmacia, Piscataway, NJ). Running condition were identical to the ones described in Pichersky et al. (1995). Enzyme eluted from DE53 in the 0.2-0.3 M KCl range, from the hydroxyapatite column in the 0.13-0.18 M sodium phosphate range, and from the MonoQ column in the 0.14-0.17 M KCl range.

Protein sequence determination Sequence analysis was performed on peptides produced from cyanogen bromide cleavage of purified BEAT as previously described (Dudareva et al., 1996). cDNA isolation and characterization For PCRTM-amplification of fragments of BEAT cDNA, the inventors synthesized several pairs of degenerate primers based on the peptide sequences. PCRTM studies were performed as previously described using the C. breweri flower cDNA library as the target (Dudareva et al., 1996). PCRTM studies using the 20-mer oligonucleotide 5'-GG (GATC) AA (TC) TT (TC) TT (TC) AT (CTA) GT (GATC) GT-3' (SEQ ID N0: 4), designed based on the peptide sequence GNFFIVV (sense orientation) (SEQ ID NO: 5), and the 23-mer oligonucleotide 5'-TT (TC) TT (TC) TC (GATC) CC (CT) TT (GATC) CCCCA (CT) TC-3' (SEQ ID N0: 6), based on the peptide sequence EWGKGEKN (antisense orientation) (SEQ ID N0: 7), gave a product of 200 nucleotides. This fragment was eluted from a 2% agarose gel, labeled, and used to screen a C. breweri floral cDNA library. Several clones were isolated and characterized by restriction enzyme digests and by sequencing. All proved to encode the same protein. The sequence of the longest clone was completely determined on both strands. A C. concinna genomic library also was screened with a C. breweri BEAT cDNA probe under high stringency conditions (65° hybridization temperature) with all other conditions being identical to the ones described in Dudareva et al., 1996.

RNA isolation and RNA gel-blot analysis Total RNA from floral tissues and petals of C. breweri and C. concinna at different stages of development was isolated and analyzed as described in Dudareva et al. (1996) and Wang et al. (1997). A 1.2 kb EcoRI fragment containing the 5'end of BEAT cDNA was used as a probe in Northern blot analysis. Total RNA levels were initially measured by spectrophotometry. For determination of tissue specificity, 7 ug of total RNA was loaded in each lane, and for time-course assays, 3 ug of total RNA was loaded. Hybridization signals were counted in a phosphoimager, and BEAT mRNA transcript levels were normalized to 18S RNA levels for graph presentation (Dudareva et al., 1996).

DNA gel blot analysis Genomic DNA (10 ug) was digested with different restriction enzymes under standard conditions, separated on 0.7% agarose gel and transferred to nitrocellulose R membrane (BioTrace NT). Hybridizations were carried out in 6 x SSC (1 x SSC is 0.15 M NaCI and 0.015 M sodium citrate), 5 x Denhardt's solution (0.1% BSA, 0.1% Ficoll 400 and 0.1% PVP 360), 0.1% SDS and 1 mM EDTA at 65°C. Blots were washed at 65°C twice with 5 x SSC and twice with 2 x SSC and autoradiographed with an intensifying screen on Kodak XAR film at-80°C (Eastman-Kodak, Rochester, NY).

Expression of BEA T in E. coli The coding region of BEAT was amplified with the forward 25-mer oligonucleotide 5'-CCATATGAATGTTACGATGCACTCC-3' (SEQ ID NO: 8), and the backward 34-mer oligonucleotide 5'-TGGATCCTTAGGAAACGTATGAAAGCAGTTGGTG-3' (SEQ ID NO: 9).

The forward oligonucleotide introduced an NdeI site at the initiating methionine ATG codon. The backward nucleotide introduced a BamHI site downstream of the stop codon, and also eliminated the single original NdeI site in BEAT cDNA which occurs 6-11 nucleotides upstream of the stop codon. The elimination of this NdeI site was

accomplished by changing the original T at the third position of the codon for tyrosine 431 to C, thus maintaining a tyrosine codon. The PCRTM-amplified 1.4 kb fragment was cloned into the NdeI-BamHI site of the expression vector pET-T7 (11a), the resulting plasmid was transferred into E. coli BL21 cells, and the expression of BEAT cDNA was induced by the addition of 0.4 mM IPTG at A600 of 0.5 with 3 hours incubation at 37°C (Wang et al., 1997). Cell cultures (25 ml) were harvested by centrifugation, resuspended in 100 mM NaCl, 50 mM Tris-HCl pH 8.0,1 mM EDTA and 1 mM PMSF, and sonicated on ice with a microtip probe for four time intervals of 30 sec each. After spinning this lysate at 12,000 x g for 5 min, soluble and insoluble fractions were assayed for enzyme activity and analyzed by SDS-PAGE followed by western blotting with anti-BEAT antibodies.

The E. coli-expressed BEAT protein was further purified by DE53 anion- exchange and MonoQ chromatography. The crude lysate was first loaded onto DEAE anion exchange column (10 ml of DE53, Whatman) preequilibrated with buffer A (50 mM Bis-Tris, pH 6.9,10% glycerol, 10 mM-mercaptoethanol) at a flow rate 0.5 ml/min. After washing off unbound materials from the column with buffer A, BEAT was eluted with step gradient of 10 mL of buffer A containing 0.1 M, 0.15 M, 0.2 M, 0.25 M, 0.35 M KC1 each. BEAT activity eluted in the 0.1-0.2 m KC1 range.

Fractions with the highest BEAT activity were pooled and dialyzed against buffer A, and loaded on a hydroxyapatite column (1 cm diameter x 10 cm, Bio-gel HT, BioRad) installed in a Pharmacia FPLC apparatus and pre-equilibrated with buffer A. The column was washed with 20 mL of buffer A and BEAT activity was eluted with 100 mL of 0-0.4 M Na phosphate with a flow rate of 0.5 ml/min. BEAT activity eluted in the 0.12-0.2 M Na phosphate range.

Next, fractions containing high levels of BEAT activity were loaded onto the FPLC anion exchange column Mono-Q (Pharmacia). After washing the column with buffer A, BEAT was eluted with a steep gradient from 0-100 mM KC1 in buffer A and a gradual linear gradient from 100-400 mM KC1 in buffer A. The C. breweri BEAT protein eluted in the 0.14-0.17 M KC1 range, as reported herein and by

Dudareva et al., (1998), but C. concinna BEAT proteins eluted in 0.11-0.115 M KC1 range.

Extraction of benzylacetatefrom E. coli cells and GC-MS analysis BL21 cells expressing BEAT and those without the pET-T7 (lla)-BEAT plasmid were grown under conditions described above. After harvesting the cells for protein purification, the spent medium (25 ml) was extracted with 5 ml of hexane, and the hexane phase removed, placed in a glass tube, and reduced to 0.2 ml by passing N2 at the opening of the tube. The samples (3 ut ouf the hexane concentrate) were analyzed by GC-MS.

Antibody production and Western blots C. breweri BEAT cDNA expressed in E. coli using the pET-T7 expression systems as described above (Studier et al., 1990). The resulting polypeptides were purified on SDS-PAGE and used to produce antibodies in rabbits (Cocalico Inc., Reamston, PA). Western blots with anti-BEAT antibodies were done as described by Dudareva et al., 1996 using a 1: 4000 dilution for the primary antibodies.

Removal of the intron from BEATgenomic clones and cloning into pET-T7for expression in E. coli Two primers which have the end of the first exon and the beginning of the second exon in antisense and sense orientations were designed. The primers were 5' -TATTTAGTACCTTTGCAGAAGGCATGAG-3' (upstream direction; SEQ ID NO: 14) and 5'-TTCTGCAAAGGTACTAAATATTCCA-3' (downstream direction; SEQ ID NO: 15). In the first step, overlapping fragments were amplified in separate PCRTM reactions. After purifying the products from the gel, a second PCRTM reaction was carried out with terminal primers only. The final PCRTM products were confirmed by sequencing. The fragment was next digested with NdeI and BamHI and ligated into the pET-T7 (11 a) expression vector also digested with NdeI and BamHI (Dudareva et al., 1998) and mobilized into E. coli Bl-21 cells.

Preparation of tissue extractsfor substratesfor enzyme assays and TLC analysis Possible endogenous substrates of BEAT were extracted from petals and leaves of C. breweri and C. concinna by grinding the tissues and incubating with methanol overnight. After spinning down the lysate, the methanol fraction was recovered and dried out by passing N2 over the samples. The remaining solid material was dissolved in ethanol. Reaction mixtures contained equal amounts (1, ug) of C. breweri BEAT or C. concinna BEAT (each purified from E. coli), 40 uL of assay buffer, 0.4 1ll of [1-14C] acetylcoenzyme A, 10 u. L of ethanol extract, and distilled water to bring the total volume up to 200 uL. Reactions were carried out at 30°C for 1 hr and 100 pL of hexane was added to extract the radiolabeled products. An aliquot (20 pL) of the organic phase was spotted onto 10 x 20 cm silica-gel 60 F254-precoated TLC plate (EM Industries, Gibbstown, NJ), and 5 aL of 5% (V/V) solution containing authentic standard was also spotted on the same plate. Plates were run and analyzed as previously described (Wang et al., 1997). Another 20 je. L of organic phase was counted in a scintillation counter. For GC analysis, enzyme reactions were carried out with 100 u. M nonradioactive acetyl-CoA for 1 hr at 30°C.

EXAMPLE 2 Temporal Variation in Emission of Benzenoid Esters The strong, sweet floral scent of C. breweri is unique in its genus and is correlated with pollination by moths, a mode of reproduction that is novel among Clarkia species (McSwain et al., 1973). The inventors have previously shown (Raguso and Pichersky, 1995) that three benzenoid esters-benzylacetate, benzylbenzoate and methylsalicylate-are constituents of the scent of C. breweri flowers. To determine the amount of these compounds emitted at different stages of floral development, the inventors performed time course headspace collections at 12-h intervals, followed by GC-MS analysis. The inventors began headspace collection with buds on the evening before they opened and ended it 5 days later.

Emission of all three esters began just before the flowers opened (10-20 % from the maximal level) and remained relatively stable for the first 12 hours after

anthesis (FIG. 1A, FIG. 1B, FIG. 1C). Emissions of benzylacetate (FIG. 1A), benzylbenzoate (FIG. 1B) and methylsalicylate (FIG. 1C) showed similar patterns over time. Emission of benzylacetate peaked on days 1,12 hours earlier than peak emission of benzylbenzoate. Emission of methylsalicylate also peaked on day 1, but was stable for the next 12 hours. Emission of all three esters declined afterwards, but remained high (50-75 % of the peak level) and relatively stable during the next 36 to 48 hours, with possibly a second minor peak on the third day after anthesis. Emission of all three esters rapidly declined after day 3, although benzylbenzoate showed a additional minor peak on day 5. During the lifespan of the flower, marked variation in ester emission between the day and night periods was not observed. Quantitatively, emission of benzylacetate was the highest, peaking at 15.8 ug/flower/12h. Benzylbenzoate and methylsalicylate were emitted at much lower levels, peaking at 2.7, ug/flowertl2h and 1.9 pg/flower/12h, respectively.

EXAMPLE 3 Localization and Quantification of Benzenoid Esters Emission from the Different Parts of the Flower To determine the specific parts of the C. breweri flowers that emit these three volatile esters, the inventors performed studies in which living flowers were modified by selectively excising floral parts, so that only one class of major floral organs (petals, stamens, pistil) remained attached to the hypanthium. The inventors then collected headspace volatiles from these modified flowers during a 24 hours period.

The data obtained were used to calculate the contribution of each floral part to the total emission of the flower (FIG. 2). These data revealed that the petals were the organs responsible for most of the emission of benzylacetate and methylsalicylate, although substantial emission of benzylacetate was also detected from stamens (one- half of the amount emitted by the petals). On the other hand, the pistil was the main source of benzylbenzoate emission, although some benzylbenzoate was also emitted by petals and stamens.

However, the emission of benzylalcohol and benzylacetate in the modified flowers was somewhat reduced. When values obtained for separate organs were added up, emission of benzylacetate totaled 60% that of the intact flower, and emission of benzylbenzoate totaled 77%. On the other hand, emission of methylsalicylate by separate organs actually exceeded (123%) that of the intact flower. These results are similar to those previously observed with several phenylpropanoid scent components of C. breweri, where the sum of (iso) eugenol emission decreased (by greater than 50%) and (iso) methyleugenol emission increased when flower parts where removed (Wang et al., 1997). Such results could mean that organs other than the emitting one are involved in controlling the flux of the pathway.

However, the most likely explanation is that such decreases and increases were brought about by the injury sustained by the flowers in these studies. It is noteworthy that emission of methylsalicylate, a compound known to be involved (together with salicylate) in the response of plant vegetative tissue to pathogen damage (Shulaev et al., 1997), increased in the injured flowers. Since salicylate is derived from the benzoic acid pathway (Yalpani et al., 1993), it is perhaps not surprising that increased synthesis of salicylate (as an intermediate in the synthesis of methylsalicylate, and possibly as end product), resulted in the concomitant decrease in the biosynthesis of benzylacetate and benzylbenzoate.

EXAMPLE 4 BEAT and SAMT Activities in Flowers BEAT and SAMTActivity in Flower Parts In many plant species, volatile benzyl esters contribute significantly to the total floral scent output (Knudsen et al., 1993). Specifically, benzylacetate, benzylbenzoate and methylsalicylate are found in the scent of many moth-pollinated flowers (Knudsen and Tollsten, 1993; Kaiser, 1993). In addition, methylsalicylate is also reported to be important in plant defense and communication (Dicke et al., 1990; Shulaev et al., 1997). However, no enzymatic activities capable of forming these products have been reported, and the pathways leading to benzoate, benzylalcohol and salicylic acid have only been partially elucidated. For example, it is known that the

benzene ring is derived from trans-cinnamic acid (Yalpani et al., 1993; Lee et al., 1995) and that benzoic acid is converted to salicylic acid by benzoic acid 2- hydroxylase (BA2H) (Leon et al., 1993).

Although the immediate biochemical step leading to benzylacetate had not previously been determined, it appeared likely that benzylacetate could be synthesized by acetylation of benzylalcohol with acetyl-CoA as the donor of the acetyl group (FIG. 3). Therefore, the inventors devised an enzymatic assay to test for benzylalcohol acetyltransferase (BEAT) activity. Crude extracts were prepared from different flower parts of flowers of different stages, incubated with benzylalcohol and [IaC] acetyl-CoA, and the product was extracted and analyzed.

Methyl salicylate is likely to result from esterification of salicylic acid with SAM as the possible methyl donor (FIG 3). Similar reactions of the S- adenosylmethionine-dependent addition of a methyl group to the carboxylic groups of proteins in animals, yeast and plants are catalyzed by enzymes collectively termed carboxyl methyltransferases (Clarke, 1992). Therefore, the inventors developed an enzymatic assay to test for salicylic acid carboxyl methyltransferase (SAMT) activity using salicylic acid and [14C] SAM.

Levels of BEAT activity in petals on day 1 of anthesis were 7-10 fold higher than in any other floral organs on per fresh weight basis (Table 2). No BEAT activity was found in vegetative tissue. SAMT activity levels, when calculated on a per fresh weight basis, were also highest in petals, although other floral parts, in particular stigma and style, had comparable levels of specific SAMT activity. Again, no activity was found in leaves (Table 2).

TABLE 2 BEATANDSAMTACTIVITIESINC. BREWERI ONDAYI OFANTHESIS Values are averages of three independent measurements. Activity values are presented both as femtomole product per second (fkat) per mg fresh wt, and as

picomole product per second (pkat) per flower. Protein concentrations in the different organs are given in Materials and Methods, and can be used to calculate specific activities per mg protein. The total weight of each class of organs in the flowers is from Pichersky et al. (1994).

Organ BEAT BEAT SAMT SAMT (total wt per Specific Activity Total Activity Specific Activity Total Activity flower) fkatimg fresh wt pkat/flower fkat/mg fresh wt pkat/flower Petal (64 mg) 317.2 22 20.3 1. 4 28.1 0.08 Sepal (22.5 mg) 33.3 0.6 0.75 0.01 1.3 0.2 0.03 0.005 Stigma (6 mg) 30.8 6.2 0.18 0.04 22.3 1.2 0.13 0.007 Style (10 mg) 32.3 5.6 0.32 0.06 13.0 1.2 0.13 0.01 Stamen (24 mg) 7.1 1.1 0.17 0.03 The inventors also calculated the total BEAT and SAMT activity levels in each class of floral organs on day 1 of anthesis (Table 2), using the mean values of 64 mg, 22.5 mg, 6 mg, 10 mg, and 24 mg for the total weight of, respectively, the petals, sepals, stigma, style and stamens of the C. breweri flower (Pichersky et al., 1994).

Since petals possess the highest levels of BEAT and SAMT specific activities per mg fresh weight among all floral organs, and they constitute slightly more than half of the total mass of the flower, it is not surprising that 90% of the total BEAT activity and 80% of the total SAMT activity in the flower are found in the petals.

The total levels of both BEAT and SAMT activities in different floral tissues on day 1 of anthesis (Table 2) correlated with the emission of benzylacetate and methylsalicylate by the same tissue (FIG. 2), with activity being highest in petals and absent in leaves. These results are very similar to those obtained for two other enzymes involved in floral scent production in C. breweri flowers, linalool synthase (LIS) and isoeugenol/eugenol 0-methyltransferase (IEMT), where strong positive

correlation was observed between levels of enzyme activities and emissions of linalool and (iso) methyleugenol from floral tissues (Pichersky et al., 1994; Wang et al., 1997). Since no BEAT or SAMT activities were found in vegetative tissue, it is unlikely that this tissue makes a significant contribution to the final step of synthesis of benzylacetate and methylsalicylate, although earlier precursors may come from such parts of the plant.

The total BEAT activity per flower is approximately 10-fold greater than the total activity of LIS (which, like BEAT, also synthesizes a major scent component, linalool), and total SAMT activity per flower is similar to that of IEMT (which, like SAMT, also synthesizes minor scent components, methyleugenol and isomethyleugenol). The total BEAT and SAMT activities (as measured in vitro) in the flowers are in fact 10-fold greater than those needed for synthesizing the emitted amounts of benzylalcohol and methylsalicylate. This observation, however, does not necessarily mean that these two enzymes are not involved in controlling the pathway. Since they as well as other enzymes may depend on a common pathway for their substrates, a theoretical"excess"of enzyme may be necessary to produce the observed amount of volatiles in the competition for substrates. In addition, a portion of these esters may not be emitted.

Temporal Variation in BEAT and SAMTActivities The total BEAT and SAMT activity levels in each class of floral organs, from 2 days prior to anthesis and up to five days after anthesis, are plotted in FIG. 4.

BEAT activity increased gradually in petals to achieve a maximum on the 4th day after anthesis and then declined by 10% from the peak level on the 5th d. In other floral organs such as stigma, style, stamens and sepals, BEAT activity remained fairly constant during flower development (FIG. 4A).

The variation in total SAMT activity in petals during the development of the flower was different from that of BEAT. Petals of young flower buds (several days before opening the flower) already possessed substantial enzymatic activity

(approximately 40% of peak level) (FIG. 4B). Maximal SAMT activity was observed in mature flower buds, one day before anthesis. In one day old flowers the level of SAMT activity was approximately 80% of peak level. SAMT activity in petals declined from the peak level during next few days and reached the initial level (equal to level in young flower buds, 40% of peak level) on the 5th day after anthesis. As with BEAT, SAMT activity levels in stigma, style, stamens and sepals were relatively low throughout flower development.

The temporal variations in levels of SAMT activity and methylsalicylate emission are similar to those observed for LIS and linalool, where the levels of enzyme activity in the petals peaked on the day of anthesis and fell afterwards, in parallel to linalool emission (Pichersky et al., 1994). On the other hand, the temporal variation in levels of BEAT activity, which shows little or no decline at the end of the lifespan of the flower (although emission of benzylacetate does decline), is similar to that observed for IEMT, except that IEMT levels peaked on day 1 of anthesis and stayed stable afterwards (Wang et al., 1997), whereas BEAT activity did not peak until the fourth day after anthesis (FIG. 4).

Together with the inventors'previous results (Pichersky et al., 1994; Dudareva et al., 1996; Wang et al., 1997) these data show the existence of at least two types of patterns for enzymes involved in scent production in C. breweri flowers. The activities of the first group of enzymes such as LIS and SAMT increase in young flowers and decline in old ones, whereas the activities of the second group of enzymes such as IEMT and BEAT increases gradually during the lifespan of the flower and remain high in old flowers.

As previously discussed (Wang et al., 1997), the causes and consequences of high levels of activity of biosynthetic enzymes in old flowers, without concomitant emission of the volatile products, are not known. Although it is possible that the biosynthetic pathways in which these enzymes participate are blocked elsewhere, another possibility which remains to be investigated is that the products produced in

the reactions catalyzed by these enzymes are required for additional processes in the flowers other than scent emission. A third possibility is that as the flower ages, substrates may be diverted to other compartments and are not accessible to the scent biosynthetic enzymes.

EXAMPLE 5 BEAT purification Previous work has shown that in C. breweri flowers, BEAT is concentrated in the petals, with others floral parts containing lesser amounts. The inventors have also shown previously that some C. breweri plants emit more benzylacetate than others (Raguso and Pichersky, 1995). The inventors therefore began their purification with petal material only, using a C. breweri line that has high BEAT activity (317 fkat/mg petal FW). Starting with 90 ml of crude petal extract, the enzyme was successively purified through a DE53 column, hydroxyapatite column, and FPLC's MonoQ column (FIG. 5). The yield was 55-65% (by activity) on each column, with a final cumulative yield of 20%. A detailed description of the purification procedure will appear elsewhere.

After MonoQ chromatography, the inventors obtained approximately 100 u. g of a protein with apparent molecular mass of 58 kD and little or no additional proteins (FIG. 5). This protein was active in catalyzing the formation of benzylacetate from benzylalcohol and acetyl CoA (FIG. 6B). It was much less active with the benzylalcohol derivatives 2'hydroxybenzylalcohol and 3'hydroxybenzylalcohol (35% and 22%, respectively), and had only residual activity with 4'hydroxybenzylalcohol (Table 3).

TABLE 3 Relative activity of C. breweri petal BEAT with benzylalcohol and related substrates.

Plant-purified enzyme E. coli-expressed enzyme Benzylalcohol 100% 100% 2'hydroxybenzylalcohol 35% 34% 3'hydroxybenzylalcohol 23% 20% 4'hydroxybenzylalcohol 5% 1% 1Set arbitrarity at 100% EXAMPLE 6 Protein Sequence Determination, Isolation and Characterization of BEAT cDNA N-terminal sequencing of this protein was unsuccessful due to a blocked N- terminus. The inventors therefore prepared several peptides by CnBr cleavage of 10 pg of purified BEAT and determined partial sequence of three of them (FIG. 7). The peptide sequences were used to construct oligonucleotides for PCR Tm amplification of a 200-nucleotide fragment of the BEAT coding sequence (see Study procedures). This fragment was in turn used to screen a C. breweri floral cDNA library (Dudareva et al., 1996). Several cDNA clones, all containing the same open reading frame of 433 codons (starting with a methionine codon), were isolated. The sequence of the longest cDNA clone is shown in FIG. 7. The clone contains a total of 1564 nucleotides, with 115 nucleotides at the 5'non-coding region and 150 nucleotides at the 3'non-coding region. That the methionine codon at position 116-118 is the initiating codon is indicated by the presence of nonsense codons in-frame upstream of it. Primer extension studies also indicate that this cDNA clone is missing only 10-13 nucleotides from its 5'end.

The protein encoded by BEAT cDNA contains all the peptide sequences determined by study (FIG. 7). The complete sequence does not share extensive

sequence similarity to any sequences of enzymatically characterized proteins currently in the databanks. It does show a limited similarity to several proteins, such as the CER2 (Negruk et al., 1996) protein of Arabidopsis and GLOSSY2 from maize (Tacke et al., 1995), believed to be involved in the elongation step of C30 to C32 in the biosynthesis of cuticular wax and which possibly interact with acetyl CoA. This region (amino acid positions 135-163, shown in black-on-white in FIG. 7) might therefore be involved in acetyl CoA interaction. The sequence of C. breweri BEAT also shows somewhat more extensive sequence identity, although still less than 50% overall, with one group of Arabidopsis ESTs (all encoding a single sequence, with limited sequence determined so far [e. g., Genbank accession number R83945]), and with an unidentified gene present on chromosome 1 [Genbank accession number AC000103] whose sequence has recently been determined through the Arabidopsis genome project.

The protein encoded by BEAT has a predicted molecular mass of 48.2 kD, considerably smaller than the 58 kD apparent molecular mass calculated from its migration in SDS-PAGE gels. Such discrepancies are not uncommon (e. g., Oh-oka et al., 1986), and may be caused in this case, at least in part, by an unusual concentration of charged residues in positions 222-247. In this region of 26 amino acids, 9 residues are negatively charged and 8 residues are positively charged, with 8 of the negatively charged amino acids clustered in the center of this short segment. Since the BEAT protein produced in the E. coli expression system (see below) has the same apparent molecular mass on SDS-PAGE as BEAT purified from petals, it is unlikely that post- translational modifications are responsible for the increase in apparent molecular mass.

EXAMPLE 7 Expression of BEAT cDNA in E. coli To further verify that the cDNA they isolated encodes BEAT, the inventors cloned it into the pET-T7 (l la) expression vector, transformed E. coli cells with the recombinant plasmid, and induced the expression of this foreign gene. E. coli

expressing this gene contained large amounts of a protein with apparent molecular mass on SDS-PAGE of 58 kD, same as the plant-purified BEAT, both in insoluble inclusion bodies (FIG. 5) and in soluble form. This protein had a very similar enzymatic activity to that of plant BEAT (FIG. 6C and Table 3). E. coli cells harboring a pET-T7 (1 la) plasmid without the BEAT coding region did not have any BEAT activity, nor did bacteria lacking this plasmid entirely. Furthermore, the spent medium in which E. coli cells expressing BEAT grew contained significant amounts of benzylacetate (2 pg/ml) (FIG. 6D), whereas bacterial cells not containing the recombinant plasmid did not (FIG. 6E). Both types of cultures produced copious amounts of indole.

EXAMPLE 8 Developmental and Tissue-Specific Expression The levels of BEAT mRNA in different parts of the flower were examined in the C. breweri line with the high BEAT activity (FIG. 8A). Highest levels of message were observed in petals, followed by style, sepals, and stamens. Very low levels were observed in the stigma, and none in leaves. These mRNA levels parallel the BEAT activity profile obtained for this line, in which highest activity is found in petals and none in the leaves (Table 4).

TABLE 4 Comparisons of BEAT activity levels between high-and low-activity plants.

BEAT Activity in"high activity"BEAT Activity in"low activity" line (fkat/mg FW) line (fkat/mg FW) Leaf 0 0 Sepal 33 20 Petal 317 24 Stigma 30 14 Style 32 40 Stamen 43 33 The inventors also examined the levels of BEAT mRNA in a second line of C. breweri. Plants in this line have a 13-fold reduction in BEAT activity in petals compared with the high-activity line (24 fkat/mg FW vs. 317 flcat/mg FW), whereas BEAT activity levels in other parts of the flower are not significantly different (Table 4). Surprisingly, BEAT mRNA levels in petals in this line were only 16% lower compared with the levels of BEAT mRNA in petals of high-activity plants (FIG. 8B; numerical data calculated from counting in a phosphoimager and normalizing to rRNA levels). The mRNA levels in other parts of the flower were also similar to those found in the high-activity line.

Flowers of C. breweri usually last 5-6 d, even when not pollinated. The steady-state level of BEAT mRNA in petals of the high-activity line were examined during the lifespan of the flower (FIG. 9). mRNA was first detected just before the flower opened, and its level was highest at anthesis. After the second day, mRNA levels declined sharply. It should be noted that although in the bud, and during the first two days after anthesis protein and mRNA levels increased in parallel, BEAT activity continued to increase and only began to decrease on day 5, whereas mRNA levels began to decline already on day 3.

EXAMPLE 9 Genetic Analysis of the Control of BEAT Expression The inventors further examined the genetic control of BEAT expression by crossing high-activity and low-activity plants and examining the segregation of 50 F2 individuals (FIG. 10). Although F plants had activity levels intermediate between the high and low levels observed in the parents it was noted that many F2 individuals had higher BEAT activity than that the high-activity parent (values for F2 individuals ranged up to 774 fkat/mg petal FW, compared with 317 fkat/mg petal FW for the high-activity parent). Nonetheless, a group of 11 individuals with low activity was clearly distinguished. A group of roughly 28 individuals could also be grouped together as"intermediate activity"plants because of their bell-shaped distribution centered around the 350 fkat mark (FIG. 10). The rest of the plants, numbering 11, were classified as"high-activity".

The inventors next examined this segregating population by performing Southern-blot analysis with a BEAT probe on parental and F2 DNA. Parental DNA was first digested with 5 different enzymes. Restriction fragment patterns with BamHI, EcoRI, EcoRV, and XbaI were identical in both parents, and differed for HindIII (see FIG. 11A for the Southern of the low-activity parent). A single band was observed with BamHI, EcoRV, and XbaI digestions (FIG. 11A), suggesting that there is a single BEAT gene locus in the C. breweri genome. Several bands were observed when the DNA was digested with EcoRI and HindIII. The coding region of BEAT has one HindII site spanning the region covered by the probe, and additional EcoRI and HindIII sites may occur in introns.

The inventors also analyzed the F individuals by DNA blots, taking advantage of the polymorphism in the HindIII restriction fragments of the two parents of the 11 F plants classified as"low activity" (FIG. 10), 10 were analyzed by Southern blots and all were homozygous for the BEAT allele of the low-activity parent (HindIII

fragments of 1.6,2.4 and 2.5 kb, in addition to two invariable fragments, see representative samples in FIG. 11A). Of the 11 Fs classified as"high activity" plants, 10 were analyzed by Southern blots. The four plants with the highest levels of activity were all homozygous for the BEAT allele of the high-activity parent (HindIII fragments of 1.1 and 1.9 kb and the two invariable fragments, see FIG. 11B).

However, of the remainder, only two were such homozygotes, and the other four, including the F2 plant (FIG. 11 B, second lane from right) with the fifth highest-level BEAT activity (588 fkat/mg FW), were heterozygous. Of the 9 F2 plants belonging to the"intermediate activity"group that were analyzed, 7 were determined to be heterozygous. One plant, with activity levels higher than the mean for the "intermediate activity"plants, was found to be homozygous for the"high activity" allele, and another plant, with activity levels lower than the mean for the"intermediate activity"plants, was found to be homozygous for the"low activity"allele.

EXAMPLE 10 Isolation and characterization of BEAT-related genes from C. concinna A combined search for C. concinna BEAT-related genes, involving the screening of a genomic library with a C. breweri BEAT probe and the PCRTM-based amplification of sequences from C. concinna using oligonucleotides specific for the beginning and end of the C. breweri BEAT gene, yielded four different genes (SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, these genes encode proteins having the sequences of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, respectively). Three of these, BEAT1 (encoding SEQ ID NO: 10), BEAT2 (encoding SEQ ID NO: 11), and BEAT3 (encoding SEQ ID NO: 12), encode proteins with high similarity (92-95% identity) to C. breweri BEAT (FIG. 12 and Table 5). A fourth gene appears to be a defective BEAT-related gene because the last third of the coding region was missing (FIG. 12). Each of the four genes contain a single intron, which varied in size among BEAT1, BEAT2 and BEAT3 from 501-533 nucleotides and was 330 nucleotides in BEAT4 (SEQ ID NO: 13). In all four genes, the intron occurred at the same position, separating the condon 193 (lysine) from

codon 194 (valine). Since a genomic clone of C. breweri BEAT has not yet been isolated, it is not known whether it too has an intron, although preliminary analysis by Southern blot, using internal probes hybridizing to internal fragments, suggest that it does.

Table 5 Similarity between BEAT1, BEAT2, BEAT3 of C. concinna and C. breweri BEAT C. breweriBEAT BEAT1 BEAT 2 BEAT3 (% of Identity) C. breweri 94. 45 95. 15 92.15 BEAT BEAT1 24/433 94.43 93.96 BEAT2 21/433 24/431 93.50 BEAT3 34/433 26/431 28/431 Comparisons of 5'and 3'non-coding regions found on BEAT1 and BEAT3 (BEAT2 was obtained by PCRTM and therefore no 5'and 3'non-coding sequences are available) indicate that those sequences in BEAT3 are most similar to the corresponding C. breweri BEAT sequences, although the protein encoded by BEAT3 is the most divergent (92% identity) to C. breweri BEAT.

EXAMPLE 11 Expression of BEAT genes and amount and activity of BEAT protein in C. concinna compared with C. breweri In C. breweri, BEAT mRNA levels varied by 2-fold among the different parts of the flower, but no mRNA was found in vegetative tissue (FIG. 13). However, BEAT protein levels were not strongly correlated with levels of mRNA, and floral tissues other than petals had substantially lower amounts of BEAT protein compared to the amount found in petals. There was a strong correlation between the levels of BEAT protein and the level of BEAT enzymatic activity. In C. concinna, on the other hand, there was a strong correlation between the level of BEAT mRNA and the

amount of BEAT protein, but there was very little BEAT activity even though BEAT protein levels in C. concinna were comparable to those found in C. breweri.

Levels of BEAT mRNA, protein and enzymatic activity were also compared in petal tissue of the two species during the lifespan of the flower (FIG. 14). Again, it was found that in C. breweri mRNA levels varied over time but that protein levels did not strongly correlated with mRNA levels but were relatively stable, and that BEAT activity level was strongly correlated with BEAT protein levels. In C. concinna, BEAT mRNA and protein levels varied little over the life span of the flower, but that BEAT enzymatic activity was much lower than expected based on the measurements of BEAT protein levels.

These results indicate that although the BEAT-like gene of C. concinna are expressed in both floral and vegetative tissues, and although some protein results from this expression (albeit in both cases at lower levels than that found for C. breweri BEAT), nevertheless little or no BEAT activity is found in crude extracts of C. concinna tissues.

EXAMPLE 12 Characterization of C. concinna BEAT proteins produced in an E. coli expression system.

In order to biochemically characterize the proteins encoded by the C. concinna BEAT genes, the inventors cloned them (after removal o the intron by PCRTM amplification) into the expression vector pET-T7 and expressed them in E. coli. The proteins were then purified in several chromatographic steps prior to analysis. Our results indicate that C. concinna BEAT1 and BEAT2 genes encode proteins with substantial activity with benzylalcohol and acetyl-CoA (Table 6). As is the case with the C. breweri BEAT, they too can acetylate other alcohols. The only significant difference between C. breweri BEAT and C. concinna BEATs is that the latter proteins have an especially strong activity with intermediate-chainlength alcohols (about 4 fold greater than C. breweri BEAT).

Table 6 Relative Activity of BEATs with Benzylalcohol and Related Substances SubstrateSubstrateC. BEAT1BEAT2BEAT 100100Benzylalcohol100 Cinnamyl alcohol 97 147 161.9 Naphthaleneethanol 57.3 138.9 93.2 2-Phenylethanol 28.3 11.4 20.7 2-Hydrozybenzylalcohol 3-Hydrozybenzylalcohol 4-Hydrozybenzylalcohol Linalool 3-cis-hexene-1-ol 102 78.7 127.8 Heptanol 143.2 498 527.7 1-Hexaconsanol 14.4 1-Triacontanol 7.0 The kinetic parameters of C. concinna BEATs were further characterized as shown in Table 7. The Kms of C. concinna BEATs were found to be similar to that of C. breweri BEAT, but their turn-over (kyat) rates were 2-3 fold lower.

Table 7 Km and Kcat of Purified BEATs from C. breweri and C. concinna Km K(sec")K/K(pM"sec") C. 1440.014399.3BEAT from Plant C. breweri BEAT 243 0.0147 60.49 BEAT1 122 0.0051 41.80 BEAT2 335 0.0074 22.09

EXAMPLE 13 Presence of benzylalcohol and benzylacetate in C. breweri and C. concinna tissues Methanolic extracts of floral and vegetative tissues of both C. breweri and C. concinna could not detect benzylacetate, even though C. breweri flowers emit up to 15 zig of benzylacetate per flower in a 24 hr period. This suggested that the benzylacetate that was made by C. breweri flowers did not accumulate in the cells but was immediately emitted. To determine the relative pools of benzylalcohol in floral and vegetative tissues of C. breweri and C. concinna, the inventors extracted tissue samples with methanol and used the extracts to carry out a reaction with acetyl-CoA and catalyzed by either C. breweri BEAT or C. concinna flowers is greater by a factor of 5 and also that the vegetative tissue of both species contains some benzylalcohol.

C. breweri BEAT was more efficient in converting benzylalcohol into benzylacetate, and both enzymes reacted with other, unidentified substrates in the methanolic extract and converted them into acetylesters.

* * * All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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