EP0858507A2 - Utilisation de synthase de linalol dans le genie genetique de la production d'odeurs - Google Patents

Utilisation de synthase de linalol dans le genie genetique de la production d'odeurs

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Publication number
EP0858507A2
EP0858507A2 EP96939462A EP96939462A EP0858507A2 EP 0858507 A2 EP0858507 A2 EP 0858507A2 EP 96939462 A EP96939462 A EP 96939462A EP 96939462 A EP96939462 A EP 96939462A EP 0858507 A2 EP0858507 A2 EP 0858507A2
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Prior art keywords
nucleic acid
sequence
acid segment
seq
linalool
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EP96939462A
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German (de)
English (en)
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Eran Pichersky
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University of Michigan
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University of Michigan
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates generally to the fields of the floral emission of monoterpenes and the production of monoterpenes by plants.
  • the present invention also relates to the field of the production of enhanced scent and taste in plants.
  • the scent emitted by such flowers is often a complex mixture of low molecular weight compounds, and the relative abundances and interactions of the constituents give the flower its particular characteristic fragrance.
  • Floral scents have been demonstrated to function as long and short-distance attractants and nectar guides to a variety of animal pollinators (reviewed in Dobson, 1993) .
  • insects are able to distinguish between complex floral scent mixtures (Dodson et al., 1969; Pellmyr, 1986).
  • fragrance Although perfumers still survey natural sources for novel fragrance compounds (Joulain, 1987; Kaiser, 1991) , this information is most often used in directing organic syntheses to imitate natural fragrances or create new combinations. There is a need, therefore, for purified synthetic enzymes that can be produced in commercial quantities for use in the manufacture of fragrance.
  • Monoterpenes are a large and diverse group of natural products. Due to their volatility, and thus their ability to be perceived at a distance, they are often involved in plant-insect interactions (Harborne, 1991; angenheim, 1994) . In addition to pollinator attraction (Dobson et al . , 1993; Knudsen et al . , 1993b), monoterpenes also play an important role in plant defense (Langenheim, 1994; Lewinsohn et al . , 1992a) or may act as semio-chemicals (Turlings et al . , 1990; Gijzen, 1993).
  • Monoterpenes are also of commercial value as essential oils for perfumery and flavoring use and as industrial raw materials (Dawson, 1994) .
  • Monoterpenes are derived from the ubiquitous isoprenoid intermediate GPP by a class of enzymes called monoterpene synthases (also termed cyclases when they catalyze the formation of cyclic products) .
  • monoterpene synthases also termed cyclases when they catalyze the formation of cyclic products
  • monoterpene synthases also termed cyclases when they catalyze the formation of cyclic products
  • monoterpene synthases from plants have been described, only a few of these enzymes have been purified to homogeneity and characterized in detail (Alonso et al . , 1992; Lewinsohn et al . , 1992b) .
  • few if any of the genes encoding these enzymes have been identified. There is an immediate need, therefore, for the identification and isolation of
  • the present invention seeks to overcome these and other drawbacks inherent in the prior art by providing a purified linalool synthase polypeptide and by further providing an isolated nucleic acid segment encoding the linalool synthase polypeptide.
  • an important embodiment of the invention may be described as an isolated nucleic acid segment comprising a nucleic acid sequence encoding a linalool synthase protein or polypeptide.
  • the nucleic acid sequence may encode an S-linalool synthase polypeptide and may more particularly encode a Clarkia breweri S-linalool synthase polypeptide.
  • the isolated nucleic acid segment of the present invention may encode the amino acid sequence disclosed herein as SEQ ID NO:2.
  • the invention may also be described as a nucleic acid segment comprising a nucleic acid sequence consisting essentially of the nucleic acid sequence of SEQ ID N0:1.
  • nucleic acid sequences may vary considerably, and still encode essentially the same polypeptide. Therefore, a nucleic acid sequence may be altered for various reasons that include, but are not limited to the use of codons that occur more frequently in the gene sequences of a particular host organism, or to insert or delete a restriction enzyme recognition site for ease in moving the particular sequence into or out of a vector, without altering the function of the polypeptide. So, while it is understood that an embodiment of the present invention is a nucleic acid segment that has the nucleic acid sequence of SEQ ID N0:1, certain variations as described are also encompassed by the present invention.
  • amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above.
  • nucleic acid sequences which may, for example, include various non-coding sequences flanking either of the 5' or 3 ' portions of the coding region or may include various internal sequences, i.e., introns, which are known to occur within genes.
  • nucleic acid segment is intended to refer to a nucleic acid molecule which has been isolated free of total cellular DNA or RNA, as the case may be, of a particular species. Therefore, a nucleic acid segment encoding a linalool synthase is intended to refer to a nucleic acid segment which contains such coding sequences yet is isolated away from total RNA or DNA of a Clarkia breweri cell.
  • nucleic acid segment includes DNA segments, whether isolated from genomic or cDNA sources or even prepared synthetically, and RNA segments which may be isolated mRNA or RNA obtained by in vi tro or in vivo transcription of a DNA segment, or a chemically synthesized RNA molecule.
  • the term also includes DNA or RNA segments which may be employed in the preparation of vectors, as well as the vectors themselves, including, for example, plasmids, cosmids, phage, viruses, and the like.
  • various types of DNA composition may be used for delivery to recipient cells in accordance with the present invention.
  • DNA segments in the form of vectors and plasmids, or linear DNA fragments, in some instances containing only the DNA element to be expressed in the plant, and the like may be employed.
  • Vectors, plasmids, cosmids, YACs (yeast artificial chromosomes) and DNA segments for use in transforming such cells will, of course, generally comprise the Lie gene.
  • These DNA constructs can further include structures such as promoters, enhancers, polylinkers, or even regulatory genes as desired.
  • the nucleic acid segments of the present invention may be positioned under the control of a promoter, and may even be positioned under the control of a recombinant promoter.
  • the promoter may be in the form of the promoter which is naturally associated with a linalool synthase gene in Clarkia breweri cells, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment or exon.
  • a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with a linalool synthase gene in its natural environment.
  • Preferred constructs will generally include a plant promoter such as the CaMV 35S 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 & Russell, 1990), ⁇ -tubulin, actin (Wang et al. , 1992), cab (Sullivan et al .
  • 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 promoters.
  • promoter that effectively directs the expression of the nucleic acid segment in the cell type chosen for expression.
  • the use of promoter and cell type combinations for protein expression is generally known to those of skill in the art of molecular biology, (for example, see Sambrook et al . (1989); Methods in Plant Molecular Biology and Biotechnology, Eds: B.R. Glick, J.E. Thompson. CRC Press, 1993; Gelvin et al. , 1990).
  • the promoters employed may be constitutive, or inducible, and can be used under the appropriate conditions to direct high level expression of the introduced nucleic acid segment, such as is advantageous in the large-scale production of recombinant proteins or peptides.
  • promoter systems contemplated for use in high-level expression include, but are not limited to, the T7 RNA polymerase promoter system described by Tabor & Richardson (1985) and the maltose binding protein-fusion protein system (Nagai & Thogersen, 1987) .
  • Constructs will also include the Lie gene 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 AgrroJbac erium tu-ne_.asciens (Bevan et al . , 1983), the terminator for the T7 transcript from the octopine synthase gene of AgrroJacterium tumefa ⁇ ciens , and the 3 ' end of the protease inhibitor I or II 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), may further be included where desired.
  • 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.
  • 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, and in floral or fruit tissue in particular, will be most preferred.
  • vectors for use in accordance with the present invention may be constructed to include the oca enhancer element.
  • This element was first identified as a 16 bp palindromic enhancer from the octopine synthase ( oca) 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 oca 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 transformation.
  • tissue-specific promoter sequences for use in accordance with the present invention.
  • the production of a transformed cell includes the introduction of an exogenous DNA segment, such as a cDNA or gene, into a recipient cell to create the transformed cell.
  • an exogenous DNA segment such as a cDNA or gene
  • the frequency of occurrence of cells receiving DNA is believed to be low.
  • it is most likely that not all recipient cells receiving DNA segments will result in a transformed cell wherein the DNA is stably integrated into the plant genome and/or expressed. Some may show only initial and transient gene expression. However, certain cells from virtually any plant species may be stably transformed, and these cells developed into transgenic plants, through the application of the techniques known in the art. There are many methods for introducing transforming DNA segments into cells, but not all are suitable for delivering DNA to plant cells.
  • Suitable methods are believed to include virtually any method by which DNA can be introduced into a plant cell, such as by Agrobacterium infection, direct delivery of DNA such as, for example, by PEG-mediated transformation of protoplasts (Omirulleh et al., 1993), by desiccation/inhibition-mediated DNA uptake, by electroporation, by agitation with silicon carbide fibers, by acceleration of DNA coated particles, etc.
  • acceleration methods are preferred and include, for example, microprojectile bombardment and the like.
  • certain cell wall-degrading enzymes such as pectin-degrading enzymes, may be employed to render the target recipient cells more susceptible to transformation by electroporation than untreated cells.
  • recipient cells are made more susceptible to transformation, by mechanical wounding.
  • friable tissues such as a suspension culture of cells, or embryogenic callus, or alternatively, one may transform immature embryos or other organized tissues directly.
  • pectolyases pectolyases
  • 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.
  • a further advantageous method for delivering transforming DNA segments to plant cells is microprojectile bombardment.
  • 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 plant cells, is that neither the isolation of protoplasts (Cristou et al . , 1988) nor the susceptibility to Agrobacterium infection is required.
  • An illustrative embodiment of a method for delivering DNA into 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 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.
  • cells in suspension are preferably concentrated on filters or solid culture medium.
  • 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.
  • 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.
  • 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.
  • TRFs trauma reduction factors
  • nucleic acid segments of the present invention may also be defined as recombinant vectors when the nucleic acid sequences disclosed and described herein are combined with, or joined to nucleic acid sequences that allow the transformation of those sequences into a host cell, and in some cases, replication of those sequences in the host cell.
  • the nucleic acid segments may be defined as a recombinant expression vector capable of expressing a linalool synthase protein or polypeptide on introduction into a host cell, or alternatively as a plant transformed with such a recombinant expression vector.
  • the vector comprises a nucleic acid sequence in accordance with SEQ ID N0:1, and the vector may further comprise the pBLUESCRIPT or pBIN19 nucleic acid sequence.
  • the present invention may, in certain embodiments be defined as a recombinant host cell comprising a nucleic acid segment that encodes a linalool synthase polypeptide.
  • the recombinant host cell may be a prokaryotic cell, such as a bacterial cell, or E. coli cell, or the recombinant host cell may be a eukaryotic cell, with a preferred eukaryotic cell being a yeast cell or a plant cell.
  • the plant cell may be a part of a plant or a substructure of a plant.
  • nucleic acid segment contained in the host cell may be positioned under the control of a promoter and further that the nucleic acid segment may be positioned in a recombinant vector.
  • the recombinant vector may also be a recombinant expression vector wherein the host cell expresses a linalool synthase polypeptide.
  • the invention may be described as a nucleic acid segment hybridizable to a nucleic acid segment comprising the sequence of SEQ ID NO:l under stringent conditions, or even as consisting essentially of the complement of SEQ ID NO:l.
  • Stringent conditions are defined as relatively low salt and ⁇ or high temperature, such as provided by 0.02M-0.15M NaCl or the equivalent at temperatures of 50°C to 70°C. Such selective conditions tolerate little, if any, mismatch between the probe and the template or target strand, and would be particularly suitable for isolating linalool synthase genes.
  • the term "complement" is used to define the strand of nucleic acid which will hybridize to the first nucleic acid sequence to form a double stranded molecule under stringent conditions.
  • Stringent conditions are those that allow hybridization between two nucleic acid sequences with a high degree of homology, but precludes hybridization of random sequences.
  • hybridization at low temperature and/or high ionic strength is termed low stringency and hybridization at high temperature and/or low ionic strength is termed high stringency.
  • the temperature and ionic strength of a desired stringency are understood to be applicable to particular probe lengths, to the length and base content of the sequences and to the presence of formamide in the hybridization mixture.
  • nucleic acid sequence will hybridize with a complementary nucleic acid sequence under high stringency conditions even though some mismatches may be present.
  • Such closely matched, but not perfectly complementary sequences are also encompassed by the present invention. For example, differences may occur through genetic code degeneracy, or by naturally occurring or man made mutations and such mismatched sequences would still be encompassed by the present claimed invention.
  • nucleic acid sequences disclosed herein will also find utility as probes or primers in nucleic acid hybridization embodiments.
  • oligonucleotides which comprise a sequence of at least 15, 20, 30, 50, 100, 200, 300, 400, 500, 600, 800, 1000, 1500 or even 2000 contiguous nucleotides which corresponds to at least a 15, 20, 30, 50, 100, 200, 300, 400, 500, 600, 800, 1000, 1500 or even 2000 nucleotide contiguous sequence of SEQ ID N0:1 or its complement will be useful as probes or primers.
  • nucleic acid segments comprising a sequence of at least 2,583 contiguous nucleotides which corresponds to a sequence of at least 2,583 contiguous nucleotides of SEQ ID NO:l or its complement, such as the 2,583 contiguous nucleotides of SEQ ID NO:l that encode the mature protein, for example will be useful for various embodiments, as will a segment comprising a sequence of at least 2,610 contiguous nucleotides which corresponds to a sequence of at least 2,610 contiguous nucleotides of SEQ ID N0:1 or its complement, such as the 2,610 nucleotide sequence encoding the entire coding region of SEQ ID NO:l, or even a nucleic acid segment comprising a sequence of at least 2,681 contiguous nucleotides which corresponds to the 2,681 nucleotide sequence of SEQ ID NO:l or its complement.
  • the invention may also be described as a method of using a nucleic acid segment encoding a linalool synthase protein or polypeptide, comprising the steps of preparing a recombinant vector in which said nucleic acid segment is positioned under the control of a promoter; introducing said recombinant vector into a host cell; culturing said host cell under conditions effective to allow expression of the encoded linalool synthase protein or polypeptide; and collecting said expressed linalool synthase protein or polypeptide.
  • the invention may also be described in certain embodiments as a method of enhancing the scent production of a plant, comprising the steps of obtaining a recombinant vector capable of expressing a nucleic acid segment encoding a linalool synthase polypeptide on introduction into a plant cell; transforming said plant with said vector; and growing said plant under conditions appropriate for expression of said nucleic acid segment.
  • the nucleic acid segment may be under the control of a tissue specific promoter/enhancer, and the tissue specific promoter/enhancer may preferably be specific for floral tissue.
  • the plant is a flowering plant, petunia, rose, carnation, etc., for example.
  • the present invention may also be described, in certain embodiments as a method of enhancing the flavor of a plant, comprising the steps of obtaining a recombinant vector capable of expressing the nucleic acid segment encoding a linalool synthase polypeptide on introduction into a plant cell; transforming said plant with said vector; and growing said plant under conditions appropriate for expression of said nucleic acid segment, and preferably wherein the nucleic acid segment is under the control of a tissue specific promoter/enhancer, and more preferably wherein the promoter/enhancer is specific for fruit or leaf tissue.
  • tissue specific promoter/enhancer examples include, but are not limited to tomato, grape and tea plants.
  • An important embodiment of the present invention is a purified linalool synthase polypeptide having a molecular weight of from about 68 to 79 kDa and a specific activity of at least about 20 pkat/mg or even a specific activity of at least about 44.1 pkat/mg, or even more preferably a specific activity of at least about 395 pkat/mg. It is understood that a 1 pkat unit is defined as 1 picomole of product formed/second.
  • a linalool synthase polypeptide composition of the present invention may be obtained by separating proteins from a flower or a flower part of a Clarkia breweri plant.
  • a method of obtaining a linalool synthase protein composition one would first obtain a protein extract by mechanical or enzymatic cellular disruption and centrifugation.
  • the further purification of such a desired protein is well known in the art and within the skill of the skilled practitioner. For example, if further purification is desired, one may pass the crude protein extract over a DEAE-cellulose column or an ion-exchange column and collect the fractions with the highest linalool synthase activity.
  • Those fractions may be identified by measuring the absorbance at 280 nm, for example (to determine the presence of polypeptides) , and assaying those fractions containing proteins for linalool synthase activity.
  • Those fractions containing the desired activity may again be further purified if so desired, by pooling those fractions and passing them over a hydroxyapatite column, for example, and again collecting fractions as before.
  • the collected fractions may be even further purified if desired, by passing those fractions over a Mono-Q column and collecting the fractions with linalool synthase activity to obtain said polypeptide.
  • the polypeptide obtained by this method may in certain preferred embodiments consist essentially of the amino acid sequence of SEQ ID NO:2.
  • a certain embodiment of the present invention is also a recombinant polypeptide comprising as a part of its amino acid sequence, a sequence according to the amino acid sequence set forth as SEQ ID NO:2, or even a recombinant polypeptide that has the amino acid sequence Of SEQ ID NO:2.
  • a certain embodiment of the invention is also an antibody immunoreactive with the polypeptides as defined immediately above.
  • the antibody may be a polyclonal antibody, or is more preferably a monoclonal antibody.
  • GPP Geranyl Pyrophosphate
  • LIS Linalool Synthase
  • LSC Liquid Scintillation
  • DTT dithiothreitol
  • GC Gas chromatography
  • LPP Linalyl pyrophosphate
  • LIS S-Linalool synthase
  • Hepes N-2- hydroxyethylpiperazine-N' -2-ethanesulfonic acid
  • Tris tris(hydroxymethyl) -aminomethane.
  • FIG. 1 The formation of S-linalool from GPP by the action of S-linalool synthase and subsequent conversion to S-linalool oxides.
  • S-Linalool synthase and monoterpene cyclases have a similar ionization first step, leading to the intermediate linalyl cation (1) .
  • S- linalool is formed by water addition in the reaction catalyzed by S-linalool synthase, whereas the bound LPP (either S- or R-LPP, depending on the particular cyclase) is isomerized and cyclized to cyclohexanoid monoterpenes by monoterpene cyclases.
  • FIG. 2 SDS-PAGE analysis of S-linalool synthase throughout purification.
  • M molecular weight markers (numbers at left indicate kDa) .
  • FIG. 3 Gel permeation chromatography of the C. breweri S-linalool synthase.
  • the Mono Q purified enzyme was separated on a Superdex 75 (Pharmacia FPLC) , and gave a molecular mass corresponding to 73 ⁇ 5 kDa by comparison with protein standards : yeast alcohol dehydrogenase (M r 150,000), bovine serum albumin (M r 67,000) , hen egg white ovalbumin (M r 45,000), bovine carbonic anhydrase (M r 29,000) and lysozy e ( ⁇ f r 14,500) .
  • yeast alcohol dehydrogenase M r 150,000
  • bovine serum albumin M r 67,000
  • hen egg white ovalbumin M r 45,000
  • bovine carbonic anhydrase M r 29,000
  • lysozy e ⁇ f r 14,500
  • FIG. 4 Capillary radio-GC analysis of the product of S- linalool synthase.
  • the tracing in A is the radioactivity response to the pentane-soluble products generated by incubating C. J reweri S-linalool synthase preparation with [ 3 H]-GPP.
  • the tracing in B is the thermal conductivity detector response to authentic geraniol and linalool standards.
  • C. breweri flowers A major component of the scent of C. breweri flowers is linalool, an acyclic monoterpene alcohol common to the floral scents of numerous other plant species (Knudsen et al . , 1993a; Kaiser, 1991).
  • C. breweri flowers also synthesize and emit two linalool oxides, for which linalool is the proposed precursor (Winterhalter et al . , 1986; Pichersky et al . , 1994).
  • the present inventor has previously observed S-linalool synthase (LIS) activity in Clarkia flower parts (Pichersky et al . , 1994).
  • This enzyme catalyzes the cation-dependent and stereoselective conversion of GPP to S-linalool (FIG. 1) .
  • LIS is both developmentally and differentially regulated in the various floral organs (Pichersky et al . , 1994) .
  • Total LIS activity per flower was highest in petals, from which most of the linalool emission occurs.
  • LIS activity per fresh weight was highest in stigma and style (i.e., the pistil), but most of the linalool produced by these tissues is converted to linalool oxides by as yet unidentified enzymes.
  • the inventors report the purification and characterization of the S-linalool synthase from stigmata of C. breweri and the isolation and characterization of the cDNA gene encoding the S-linalool synthase.
  • the minor aroma constituents included linalool oxide (cis-furanoid) , benzyl benzoate, eugenol, methyl salicylate and vanillin.
  • isoeugenol, methyleugenol, methylisoeugenol and veratraldehyde were detected in all flowers from line 2 but were absent in plants from line 1.
  • These 12 compounds fall into two groups, monoterpenes and aromatic compounds with a benzene skeleton.
  • abundant headspace compounds were collected from single, living flowers of C. breweri . No volatile compounds were detected from similar amounts of floral tissues of the closely related species, C. concinna , nor from other Clarkia species.
  • the furanoid linalool oxide was emitted at much lower level, peaking at 2.2 ⁇ g/g/12 h. Emission gradually declines after D2 until approximately 24 h after pollination (D3-5) , when the flowers drastically decrease their monoterpene output. This cessation coincides with the general senescence of the flower. During the lifespan of the flower, marked variation in monoterpene emission between the day and night periods was not observed. Headspace collections made at 6 h intervals also failed to detect such changes. Diurnal variation has been previously described in some species (Matile et al . , 1988; Overland, 1960) but not in others (Loughrin et al. , 1990; Loughrin et al . , 1991).
  • Emission of linalool per fresh weight is actually higher in the style than in petals, but because the total fresh weight of the style accounts for only 10% of the total mass of the flower while the petals account for 40%, the petal contribution is predominant.
  • the style is responsible for nearly 100% of emission of the linalool oxides.
  • Sepals, hypanthium and ovaries did not emit monoterpenes.
  • C. concinna whose flowers emit monoterpenes at a level of 0.1% compared with C. breweri (per flower) , that emission occurs only from styles.
  • the result closely approximated the complete floral scent both qualitatively and quantitatively, indicating that each part is autonomous, and therefore synthesis of scent components must occur at the site of emission.
  • Determination of the level of the monoterpenes found in the flower and bud tissue indicates that there is a substantial pool of these compounds in the tissue. For example, at the time of peak emission (D2) , the amount of linalool in the tissue is approximately equal to 10% of the total emitted over the corresponding 12 h period. Similar results were found for the linalool oxides. The observations that large pools of these monoterpenes exist within the tissues and that the changes in intracellular monoterpenes concentration parallel those of monoterpene emission (as does enzyme activity) , suggest that these compounds are not sequestered for long as free monoterpenes inside the cell prior to the onset of emission.
  • LIS activity was assayed by combining crude extract with 3 H-GPP (or other substrates) in a buffer containing co-factors. LIS activity was monitored by counting the radioactive linalool extracted from the assay buffer with hexane (GPP is not soluble in hexane) in a scintillation counter. Radio-GC analyses (Croteau et al . , 1990) were also undertaken because GPP could be enzymatically hydrolyzed by non-specific phosphatases to produce geraniol, or the tissue might contain other monoterpene synthases, and geraniol or the other monoterpenes that may be produced are also soluble in the organic phase. The studies indicated that crude extracts of open C.
  • GPP GPP
  • the level of LIS activity is also positively correlated with the rates of linalool emission. Both linalool emission and LIS activity in the petals peak at the D1-D2 period, and as LIS activity decreases afterwards, so does linalool emission. There is some low level of LIS activity in petals of unopened flowers, and some linalool is made in the buds, but apparently not in sufficient amounts to be volatilized. Stigmata and pistils displayed relatively low levels of linalool emission (which is nonetheless also positively correlated with LIS activity) but substantial emission of linalool oxides. Taken together with the observation of high levels of LIS activity in stigmata and pistils, it is contemplated that most of the linalool produced in these organs is converted into linalool oxides.
  • concinna This finding raises the question of the function of the pathway in non-scented plants.
  • linalool production in vegetative tissue in response to insect damage has been observed (Turlings et al . , 1992), and it is contemplated that this compound may be used by the plants in such a response. It is contemplated, therefore, that the genes and proteins of the present invention may be useful in the protection of plants from insect damage.
  • a chromatographical purification protocol was developed that employed anion exchange (DE52) column, HAP column, and another ion-exchange column, FPLC's Mono-Q.
  • a crude extract was prepared from 50 g of stigma tissue (derived from about 20,000 stigmata). The stigmata was used because they have the highest specific activity of LIS (subsequently, LIS was also partially purified from petals, and it appears to be an identical protein) .
  • the fraction containing the peak LIS activity had a single protein, as judged by SDS-PAGE and silver staining.
  • the LIS protein was calculated to have a molecular weight of app.
  • LIS activity elutes at about 70 kDa, indicating that LIS is active as a monomer.
  • N- terminal sequencing of LIS eluted from Mono-Q gave a clear sequence (with initial yield >50%) indicating no heterogeneity in the purified enzyme (22 residues were determined) . In addition, the sequences of several internal fragments were determined.
  • mRNA was isolated from petals and stigmata and cDNA expression libraries were prepared in the lambda-ZaplI vector (Stratagene, Inc.) Several peptide sequences were used to construct oligonucleotides for PCR (polymerase chain reaction) using DNA obtained from the cDNA libraries. Fragments in the expected size range were obtained and analyzed.
  • the identity of the clones is determined in several ways.
  • the oligonucleotide PCR probes are used as sequencing primers to determine the nucleotide sequence of adjacent regions in the clones. Because only parts of the amino acid sequences which had been obtained are used in the design of the oligonucleotides (7-8 residues out of 20-25 residues per peptide sequence) , complete concordance of the predicted amino acid sequence obtained from the nucleotide sequence of the clones with the peptide sequences is a strong validation of the clones. Clones that have the correct sequences are further tested by RFLP mapping (McGrath et al . , 1993a; McGrath et al .
  • F- ⁇ hybrids between C. breweri and C. concinna are used to obtain F 2 s and F ⁇ X C. concinna backcross progeny. These plants are analyzed for emission of linalool and other volatiles. The trait of linalool emission is somewhat quantitative, but two distinct classes of strong emitters and non-emitters are clearly evident.
  • concinna stigma are not believed to be due to changes in turn-over number or other kinetic parameters of the enzyme.
  • these differences are strongly correlated with the differences in the amount of protein.
  • C. breweri has evolved the ability to make (and emit) greater amounts of monoterpenes by increasing the level of Lia gene transcription and/or increasing translational efficiency.
  • Expression levels of the Lia gene in stigma and petals in the two species may be determined by first determining the steady-state level of the mRNA with the Northern blotting technique, using the Lie cDNA clone as a probe, and also by primer extension, a technique that also allows the determination of the start of transcription (Kellman et al . , 1990; Kellman et al . , 1993; Piechulla et al . , 1991). Run-on transcription assays may also be used to measures rates of transcription (Giuliana et al . , 1988b). A correlation is then sought between transcription rates and steady-state mRNA levels, and between steady-state mRNA and LIS activity levels.
  • nucleic acid segments of the present invention may also be used to isolate and characterize the Lia genes from various tissues in both species by screening genomic libraries constructed in the lambda vector EMBL3.
  • the promoter region of the C. breweri Lia gene may now be characterized by in vivo methods, such as reporter gene constructs, for example, and used for tissue specific expression of foreign genes.
  • the present invention comprises an antibody that is immunoreactive with a linalool synthase polypeptide.
  • An antibody can be a polyclonal or a monoclonal antibody and is preferably a monoclonal antibody.
  • Means for preparing and characterizing antibodies are well known in the art (See, e. ⁇ .. Antibodies "A Laboratory Manual , E. Howell and D. Lane, Cold Spring Harbor Laboratory, 1988) .
  • spleen cells are removed and fused, using a standard fusion protocol (see, e . g. , The Cold Spring Harbor Manual for Hybridoma Development, incorporated herein by reference) with plasmacytoma cells to produce hybridomas secreting monoclonal antibodies against LIS.
  • Hybridomas which produce monoclonal antibodies to the selected antigens are identified using standard techniques, such as ELISA and Western blot methods.
  • monoclonal antibodies specific to the particular LIS enzyme may be utilized in immunoabsorbent protocols to purify native or recombinant LIS enzyme species or variants thereof.
  • both poly- and monoclonal antibodies against LIS may be used in a variety of embodiments.
  • they may be employed in antibody cloning protocols to obtain cDNAs or genes encoding LIS or related proteins. They may also be used in inhibition studies to analyze the effects of LIS in particular cells or tissue types.
  • a particularly useful application of such antibodies is in purifying native or recombinant
  • LIS for example, using an antibody affinity column, or screening for Lia expressing cells.
  • the operation of all such immunological techniques will be known to those of skill in the art in light of the present disclosure.
  • Modification and changes may be made in the structure of the encoded polypeptides used in the vectors and nucleic acid segments of the present invention and still obtain a functional molecule that encodes a protein or peptide with desirable characteristics.
  • the following is a discussion based upon changing the amino acids of a protein to create an equivalent, or even an improved, second-generation molecule.
  • the amino acid changes may be achieved by changing the codons of the DNA sequence, according to the following codon table:
  • 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, 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 sequence substitutions can be made in a protein sequence, and, of course, 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 peptide sequences of the linalool synthase proteins, or corresponding DNA sequences which encode said proteins without appreciable loss of their biological utility or activity.
  • 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, incorporate herein by reference) . 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 (-0.8) ; tryptophan (-0.9) ; tyrosine (-1.3) ; proiine (-1.6) ; histidine (-3.2) ; glutamate (-3.5) ; glutamine (-3.5) ; aspartate (-3.5) ; asparagine (-3.5) ; lysine (-3.9) ; and arginine (-4.5) .
  • 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.
  • 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.
  • an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein.
  • substitution of amino acids whose hydrophilicity values are within ⁇ 2 is preferred, those which are within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
  • amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Exemplary substitutions which 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.
  • 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, for example, 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.
  • 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 of site-specific mutagenesis is well known in the art, as exemplified by various publications.
  • the technique may employ a phage vector which exists in both a single stranded and double stranded form.
  • Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage. These phage are readily 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 plasmid to a phage.
  • site-directed mutagenesis in accordance herewith is performed by first obtaining a single- stranded vector or melting apart of two strands of a double stranded vector which includes within its sequence a DNA sequence which encodes the desired peptide.
  • An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically. This primer is then annealed with the single-stranded vector, 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.
  • DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment
  • sequence variants of the selected peptide-encoding DNA segments 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 peptides and the DNA sequences encoding them may be obtained.
  • recombinant vectors encoding the desired peptide sequence may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.
  • Volatile monoterpenoid production in individual flowers of four separate plants was monitored over a 7 d 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 h intervals, corresponding to the dark and light periods in the growth chamber. All flowers were hand-pollinated with a cotton swab on the evening of day 3 after anthesis in order to simulate the typical life cycle of a C. breweri flower.
  • the specific floral parts responsible for scent emission were determined and the emission levels were quantified by collection of headspace volatiles 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 style.
  • headspace collections were taken over 6 and 12 hr intervals.
  • a crude protein extract was prepared by macerating flower parts in a microcentrifuge tube in the presence of ice-cold buffer (10 volumes:fresh weight) containing 50 mM BisTris-HCl pH 6.9, 10 mM DTT, 5 mM Na-S 2 0 5 , 1% (w/v) PVP-40 and 10% (v/v) glycerol. The slurry was centrifuged for 10 min and the supernatant transferred to a new tube. For each time point, extracts of 3 to 5 flowers from 3 different plants were combined.
  • Linalool synthase activity was assayed by diluting 10 ⁇ L of crude extract (1.5-3 ⁇ g protein) into 80 ⁇ L of assay buffer (50 mM potassium HEPES pH 7.8, 5 mM DTT, 5 mM sodium metabisulfite, 10% (v/v) glycerol, 20 mM MgCl 2 and 5 mM MnCl 2 [Lewinsohn et al., 1991]), adding 10 ⁇ L of [1- 3 H] -GPP to final concentration 16 ⁇ M, at 150 mCi/mol [substrate synthesized according to Croteau and Cane (1985)], and overlaying the mixture with either pentane or hexane to trap volatile metabolites.
  • assay buffer 50 mM potassium HEPES pH 7.8, 5 mM DTT, 5 mM sodium metabisulfite, 10% (v/v) glycerol, 20 mM MgCl 2 and 5 mM Mn
  • the tube was then vortexed briefly and incubated at 20°C for 1 h.
  • Appropriate controls included the omission of crude extract, the use of boiled crude extracts, the omission of substrate and/or cations, and the substitution of other potential substrates such as (3R)- and (3S) - [1- 3 H] - linalyl pyrophosphate.
  • the tube was vortexed again and the amount of the radioactive linalool product, which partitioned into the organic phase, was determined by an LSC of an aliquot.
  • Radio-GC analysis (Croteau and Satterwhite, 1990) of labelled products in the organic phase was also undertaken to examine the formation of geraniol (liberated from the substrate by phosphatases) and to determine whether other monoterpene synthases were present in the extracts.
  • These radio-GC analyses showed that no floral tissue contained detectable monoterpene synthase activity other than LIS (linalool > 95% of product by coincidence of radioactivity with authentic standard) , and that only crude extracts from mature pollen grains contained appreciable phosphatase activity. Because of the presence of phosphatases in the pollen grains, anther tapeta were routinely excluded in the preparation of subsequent cell-free extracts.
  • the present inventor has previously shown (Raguso and Pichersky, 1995) that three monoterpenes - linalool, furanoid linalool oxide and pyranoid linalool oxide - are constituents of the scent of C. breweri flowers.
  • time-course headspace collections were performed at 12 h intervals, followed by GC-MS analysis. Headspace collection was performed with buds on the evening before they opened and ended 6 d later, after flowers had been pollinated and then wilted and abscised.
  • C. concinna a close relative of C. breweri, has flowers which are smaller and which do not emit a detectable scent.
  • the levels of emission of these monoterpenes in floral organs of C. concinna (Tables I,II) were then examined. The results indicate that volatile emission occurs only in pistils.
  • LIS activity was found in several parts of the flowers. At the peak of LIS activity, at Days 1-2, total LIS activity was similar in petals and pistil (stigma + style) . However, stigmata possessed the highest level of LIS activity per fresh weight, followed by style tissue (with 25-35% of the specific activity found in stigma) , petals (20% of stigma LIS specific activity at peak time) and stamens. None of the remaining floral parts - sepals, hypanthium and ovaries - were found to contain any LIS activity. The vegetative parts of the plant were also devoid of LIS activity.
  • LIS activity varied during the lifespan of the flower in concert with the levels of linalool emission and its concentration in floral tissue. Maximal LIS activity was observed during Days 1-2. When flowers were pollinated on Day 3, LIS activity decreased by 90% in the stigma and 50% in the petals within 48 h, similar to the reduction in monoterpene emission following pollination. However, when pollination did not occur,
  • LIS activity in extracts of C. concinna flower parts of Day 2 flowers was examined. Consistent with the emission data (Tables I,II), LIS activity was detected only in the stigma, which had a level of activity of 2.8 fkat/stigma (and per flower) and 4.6 fkat/g fresh weight. These levels are respectively 0.3% of the LIS activity per stigma (0.01% per flower) and 3% per fresh weight of activity in C. breweri stigmata of the equivalent stage.
  • 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 (MacSwain et al. , 1973). Emission of the monoterpene components of the scent begins as soon as the flowers are open and reaches a peak on Day 2. During the lifespan of the flower, marked variation in monoterpene emission between the day and night periods was not observed. It is possible that temporary, but substantial, increases or decreases in emission were missed because of their short duration, but headspace collections made at 6 h intervals also failed to detect such changes, and samples did not vary by more than a factor of 1.5.
  • the highest and second highest levels of LIS activity per fresh weight were found in the stigma and style, respectively.
  • the pistil is the only part of the flower (excluding the ovaries) that continues to increase in size and weight after the flower opens, but its specific LIS activity does not decrease, indicating that additional LIS activity accrues there, at least during the first few days after anthesis.
  • the petals constitute the bulk of the LIS-containing floral tissue (Table III) , and they possess a similar or even higher total amount of LIS activity compared with the pistil.
  • Each flower part that emits linalool or linalool oxides - petals, pistils and stamens - also contains significant LIS activity, whereas flower parts that do not contain LIS activity do not contain or emit these monoterpenes.
  • the levels of LIS activity in the different parts of the flower throughout the lifespan of the flower are also positively correlated with the rates of emission of linalool and the two linalool oxides.
  • Both monoterpene emission and LIS activity in the petals, pistil and stamens peak during the first two days after anthesis, and as LIS activity decreases afterwards (especially after the flower has been pollinated) , so do linalool and linalool oxides emissions.
  • buds of later stages contain appreciable amounts of LIS activity but they do not accumulate or emit these monoterpenes. It is likely that earlier steps in the pathway are not yet operating in the buds, either for lack of other enzymes or because of sequestration of enzymes and/or substrates in different subcellular compartments.
  • monoterpenes may be synthesized in buds of later stages but may be rapidly converted to other compounds or derivatives.
  • the pistil displayed relatively low levels of linalool emission but substantial emission of linalool oxides (Table 2) .
  • Table 2 The pistil displayed relatively low levels of linalool emission but substantial emission of linalool oxides.
  • Table 2 The pistil displayed relatively low levels of linalool emission but substantial emission of linalool oxides.
  • the conclusion is inescapable that most of the linalool produced in these organs is converted into linalool oxides.
  • Winterhalter et al. (1986) presented evidence that, in papaya tissues, the linalool oxides are synthesized from linalool via 6,7-epoxylinalool as an intermediate (FIG. 1) . They further showed that the conversion of linalool to 6,7-epoxylinalool was enzymatically catalyzed, although a specific enzyme was not identified.
  • Crude protein extracts were prepared by homogenizing freshly excised stigmata in a chilled mortar in the presence of ice-cold buffer (10:1 (v/w) buffer:tissue) containing 50 mM potassium BisTris, pH 6.9, 10 mM dithiothreitol, 5 mM Na 2 S 2 0 5 , 1% (w/v) polyvinylpyrrolidone (Sigma, PVP-40) and 10% (v/v) glycerol. The slurry was passed through miracloth and centrifuged for 10 min at 12,000 g to produce a supernatant that contained the bulk of the LIS activity.
  • ice-cold buffer (10:1 (v/w) buffer:tissue) containing 50 mM potassium BisTris, pH 6.9, 10 mM dithiothreitol, 5 mM Na 2 S 2 0 5 , 1% (w/v) polyvinylpyrrolidone (Sigma, PVP-40) and 10% (v
  • the crude extract (100 ml) was loaded onto a DEAE- cellulose (0.7 cm diam. x 6 cm) column (Whatman, DE52) that was pre-equilibrated with a solution containing 50 mM BisTris, pH 6.9, 10% glycerol and 10 mM DTT (buffer A) at a flow rate of about 1 ml/min.
  • the column was washed with 50 ml of buffer A followed by an additional 20 ml of buffer A containing 150 mM KC1.
  • LIS activity was then eluted with 20 ml of buffer A containing 250 mM KCl .
  • the column was washed with 20 ml buffer A and then eluted using a linear gradient (100 ml) from 0 to 200 mM Na-phosphate in buffer A.
  • the fractions containing LIS activity, eluting at about 100 mM Na-phosphate, were pooled (10 to 16 ml) and loaded on a pre-packed Pharmacia Mono-Q HR 5/5 FPLC column equilibrated with buffer A.
  • a 20-ml wash with buffer A a steep linear gradient from 0 to 100 mM KCl in buffer A (10 ml) was applied, followed by a more gradual linear gradient from 100 to 400 mM KCl in buffer A (100 ml, 0.25 ml/min) .
  • the enzyme consistently eluted at about 220 mM KCl. After dialysis to assay conditions, the enzyme at this level of purity was used for characterization.
  • a Superdex 75 Hi Load 16/60 column (Pharmacia FPLC) was employed to determine the native molecular mass of the C. breweri linalool synthase. Hydroxyapatite- purified linalool synthase (1 ml, 44.1 pkat/mg protein) was loaded on the Superdex 75 column and eluted with buffer A containing 5 mM DTT, using a flow rate of 1 ml/min. Two-ml fractions were collected. Protein standards of known molecular mass (Sigma, St. Louis, MO) were used for calibration.
  • the molecular weight marker proteins were from BioRad (Richmond, CA) .
  • LIS activity was determined by mixing 10 ⁇ l of the enzyme sample with 12 ⁇ M [1- 3 H] -GPP (s.a. 150 Ci/mol) in 100 ⁇ l of assay buffer (50 mM Hepes-KOH, pH 7.8, 10 mM DTT, 5 mM Na-.S 2 0 5 , 10% (v/v) glycerol, 20 mM MgCl 2 and 5 mM MnCl 2 (Lewinsohn et al . , 1991) and, as an overlay to trap volatiles, either 1 ml hexane (for scintillation counting) or 2 ml of pentane (for radio-GC analysis) .
  • assay buffer 50 mM Hepes-KOH, pH 7.8, 10 mM DTT, 5 mM Na-.S 2 0 5 , 10% (v/v) glycerol, 20 mM MgCl 2 and 5 mM MnCl 2 (Lewins
  • the mixture was then vortexed briefly, centrifuged to separate phases (5 sec. Eppendorf) and incubated at 20°C without shaking for up to 1 h (higher temperatures resulted in elevated rates of chemical conversion of GPP to various alcohols, including linalool) . After incubation, the assay mixture was briefly mixed again to extract non-polar products into the organic layer.
  • the 2 ml pentane extract containing the enzymatically formed 3 H-labeled products was removed, dried by passage through MgS0 4 , and internal standards were added (10 ⁇ g each of linalool and geraniol; nerol was also included in initial experiments) .
  • the mixture was then concentrated to -10 ⁇ l under a gentle stream of nitrogen, and an aliquot (2-3 ⁇ l) was injected into a Gow-Mac 550P radio gas chromatograph fitted with a 0.125 in x 12 ft stainless steel column loaded with AT-1000 (Gas Chrom Q, Alltech) , attached to a Packard 894 gas proportional counter.
  • the initial column temperature was 170°C and, following a 5 min hold, was programmed at 5°C/min to 220°C, using He at a flow rate of 50 ml/min.
  • Control incubations included the omission of enzyme extract, the use of boiled enzyme extract, the omission of substrate, and the omission of divalent cations. All of these controls produced negligible radioactivity in the hexane phase of the enzyme assay mixtures.
  • Protein was determined by the method of Bradford (Bradford, 1976) , using bovine serum albumin as standard.
  • the enantiomeric composition of linalool samples was determined on a fused silica capillary column (0.25 mm internal diam. x 30 m) coated with a 0.25 ⁇ m film of ⁇ - cyclodextrin (J & W Scientific, Cyclodex B) , operated at an initial temperature of 75° C for 15 min followed by a rise of 5°C/min to 200° C. H 2 at a head pressure of 13.5 psi was used as carrier gas (Alonso et al . , 1992) .
  • Flower stigmata were used as the enzyme source, as they contained very high enzyme activity levels (147 fkat/mg Fr.Wt.) (Pichersky et al . , 1994) (See Example 1) .
  • a representative purification scheme for the C. breweri S-linalool synthase is shown in Table 5.
  • 10 g Fr.Wt. of freshly excised stigmata gave 140 ⁇ g of pure linalool synthase following the initial DEAE-cellulose and hydroxyapatite chromatography steps (to remove low molecular weight pigments and phenolic materials that severely interfered with later FPLC steps) , and Mono Q chromatography (FIG. 2) .
  • the native molecular mass of linalool synthase activity was determined to be about 73 ⁇ 5 KDa based on co-elution with bovine serum albumin on gel permeation chromatography and comparison of the elution volume to those of known proteins (FIG. 3) . Based on a subunit mass of 79 KDa determined by SDS-PAGE (FIG. 2) and a native molecular size of 73 KDa indicated by gel permeation chromatography (FIG. 3) , linalool synthase is thought to be a monomer of 76 ⁇ 3 KDa.
  • the preparations were devoid of other monoterpene synthase activities or GPP phosphohydrolase activity as evidenced by the absence of [ 3 H] -geraniol in the reaction products.
  • C. breweri leaves contained only GPP phosphohydrolase activity, as leaf- derived cell-free extracts produced only geraniol.
  • S-linalool synthase requires a divalent metal ion for activity.
  • a turnover number of 31.6 sec "1 was calculated for LIS under saturating conditions, indicating that the enzyme is much faster than other monoterpene synthases examined so far (Alonso et al. , 1992; Alonso et al . , 1993) .
  • a broad pH optimum of 7.4 was calculated for the enzyme, with the rates at pH 6.4 and 8.4 being -80% of the optimal. This optimal pH range is similar to those determined for other monoterpene synthases (Alonso et al . , 1992; Lewinsohn et al . , 1992; Alonso et al . , 1993). Similar rates were observed while substituting Hepes buffer with Tris-HCl.
  • the enzyme is unstable in the absence of DTT (>95% loss over 2 h at 4 °C) , and its later addition does not result in the regaining of activity.
  • the enzyme is very stable to vigorous vortexing in the presence of non-polar organic solvents such as hexane and pentane.
  • the starting material was 10 g of excised stigmata (approx. 2500-300 stigmata) .
  • Vasil, V., Clancy, M. Ferl, R.J., Vasil, I.K., Hannah, L.C. (1989) Plant Physiol. 91:1575-1579.
  • GCA AGA TCT TTC
  • GCT ACC AAG AAT CTT
  • GAG AAA ATA TTA
  • GCA ACA GGA 1251 Ala Arg Ser Phe Ala Thr Lys Asn Leu Glu Lys He Leu Ala Thr Gly
  • MOLECULE TYPE protein 5

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  • General Health & Medical Sciences (AREA)
  • Plant Pathology (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Insects & Arthropods (AREA)
  • Pest Control & Pesticides (AREA)
  • Medicinal Chemistry (AREA)
  • Nutrition Science (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

L'invention porte sur un polypeptide purifié de synthase de S-linalol à partir de Clarkia breweri, représentant le polypeptide recombiné et les séquences d'acide nucléique codant ledit polypeptide. L'invention concerne également des anticorps immunoréactifs au peptide purifié et aux versions recombinées dudit polypeptide. L'invention concerne enfin des procédés utilisant lesdites séquences d'acide nucléique, et des procédés de stimulation de l'odeur et du goût de plantes exprimant lesdites séquences d'acide nucléique.
EP96939462A 1995-10-12 1996-10-15 Utilisation de synthase de linalol dans le genie genetique de la production d'odeurs Withdrawn EP0858507A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US514695P 1995-10-12 1995-10-12
US5146P 1995-10-12
PCT/US1996/016482 WO1997015584A2 (fr) 1995-10-12 1996-10-15 Utilisation de synthase de linalol dans le genie genetique de la production d'odeurs

Publications (1)

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EP0858507A2 true EP0858507A2 (fr) 1998-08-19

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EP (1) EP0858507A2 (fr)
AU (1) AU7663296A (fr)
WO (1) WO1997015584A2 (fr)

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AU7377298A (en) * 1997-05-08 1998-11-27 Regents Of The University Of Michigan, The Methods and compositions for use of (iso)eugenol methyltransferase
WO1999023226A1 (fr) * 1997-10-30 1999-05-14 The Regents Of The University Of Michigan Procedes et compositions pour l'utilisation de benzylalcool acetyl transferase
AR022383A1 (es) 1998-09-18 2002-09-04 Univ Kentucky Res Found Sintasas
US7129393B1 (en) 1999-02-22 2006-10-31 Yissum Research Development Company Of The Hebrew University Transgenic plants and method for transforming carnations
AU2687000A (en) * 1999-02-22 2000-09-14 Yissum Research Development Company Of The Hebrew University Of Jerusalem Transgenic plants and method for transforming carnations
EP1130104A1 (fr) * 2000-02-16 2001-09-05 Stichting Dienst Landbouwkundig Onderzoek Réduction de la dégradation des produits végétales in planta
EP1231273A1 (fr) * 2001-02-12 2002-08-14 Plant Research International B.V. Terpene synthase/cyclase et olefin synthase et leur utilisation
CA2441594A1 (fr) * 2001-03-22 2002-10-03 Scentgene Pollination Ltd. Procede pour accroitre l'entomophilie
ES2324508B1 (es) * 2006-12-27 2010-05-31 Consejo Sup. De Invest. Cientificas Mejora del contenido aromatico de vinos y otras bebidas alcoholicas mediante la utilizacion de microorganismos que, durante la fermentacion, producen monoterpeno sintasas.
JP5956252B2 (ja) * 2011-06-10 2016-07-27 サントリーホールディングス株式会社 リナロール合成酵素をコードするポリヌクレオチドおよびその用途
CN110452918B (zh) * 2019-08-30 2021-03-09 福建农林大学 一种创制高香型矮牵牛新种质的分子方法

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GB9021923D0 (en) * 1990-10-09 1990-11-21 Ici Plc Dna,dna constructs,cells and plants derived therefrom
WO1992016611A1 (fr) * 1991-03-12 1992-10-01 Pernod-Ricard Procede de fermentation alcoolique pour obtenir des aromes de type muscat
WO1993007257A2 (fr) * 1991-10-04 1993-04-15 Smart Plants International, Inc. Sequences de transcription a specificite tissulaire et developpement regule et leur utilisations
CA2118071C (fr) * 1993-10-28 2004-09-14 Rodney B. Croteau Synthase du limonene codant pour l'adn qui provient de mentha spicata

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See references of WO9715584A2 *

Also Published As

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WO1997015584A2 (fr) 1997-05-01
AU7663296A (en) 1997-05-15
WO1997015584A3 (fr) 1997-09-25

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