Monoterpene synthases from grand fir (Abies grandis)

Details Monoterpene synthases from grand fir (Abies grandis) Monoterpene synthases from grand fir (Abies grandis)

1999-07-26

2002-08-06

Bui, Phuong T. (Department: 1638)

Chemistry: molecular biology and microbiology

Plant cell or cell line, per se ; composition thereof;...

Plant cell or cell line, per se, contains exogenous or...

C435S006120, C435S069100, C435S183000, C435S410000, C435S252300, C435S320100, C530S370000, C536S023600, C536S023200, C800S278000, C424S094100, C424S195110

Reexamination Certificate

active

06429014

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to nucleic acid sequences which code for monoterpene synthases from gymnosperm plant species, in particular from Grand fir (
Abies grandis
), including (−)-camphene synthase, (−)-&bgr;-phellandrene synthase, terpinolene synthase, (−)-limonene/(−)-&agr;-pinene synthase, limonene synthase, myrcene synthase, and pinene synthase, to vectors containing the sequences, to host cells containing the sequences, to plant seeds expressing the sequences and to methods of producing recombinant monoterpene synthases and their mutants.
BACKGROUND OF THE INVENTION
Chemical defense of conifer trees against bark beetles and their associated fungal pathogens relies primarily upon constitutive and inducible oleoresin biosynthesis (Johnson, M. A., and Croteau, R. (1987) in
Ecology and Metabolism of Plant Lipids
(Fuller, G., and Nes, W. D., eds.) pp. 76-91, American Chemical Society Symposium Series 325, Washington, D.C.; Gijzen, M., Lewinsohn, E., Savage, T. J., and Croteau, R. B. (1993) in
Bioactive Volatile Compounds from Plants
(Teranishi, R., Buttery, R. G., and Sugisawa, H., eds.) pp. 8-22, American Chemical Society Symposium Series 525, Washington, D.C.). This defensive secretion is a complex mixture of monoterpene and sesquiterpene olefins (turpentine) and diterpene resin acids (rosin) that is synthesized constitutively in the epithelial cells of specialized structures, such as resin ducts and blisters or, in the case of induced oleoresin formation, in undifferentiated cells surrounding wound sites (Lewinsohn, E., Gijzen, M., Savage, T. J., and Croteau, R. (1991)
Plant Physiol.
96:38-43). The volatile fraction of conifer oleoresin, which is toxic to both bark beetles and their fungal associates (Raffa, K. F., Berryman, A. A., Simasko, J., Teal, W., and Wong, B. L. (1985) Environ. Entomol. 14:552-556), may consist of up to 30 different monoterpenes (Lewinsohn, E., Savage, T. J., Gijzen, M., and Croteau, R. (1993)
Phytochem. Anal.
4:220-225), including acyclic types (e.g., myrcene), monocyclic types (e.g., limonene) and bicyclic types (e.g., pinenes) (FIG.
1
). Although the oleoresin is toxic, many bark beetle species nevertheless employ turpentine volatiles in host selection and can convert various monoterpene components into aggregation or sex pheromones to promote coordinated mass attack of the host (Gijzen, M., Lewinsohn, E., Savage, T. J., and Croteau, R. B. (1993) in
Bioactive Volatile Compounds from Plants
(Teranishi, R., Buttery, R. G., and Sugisawa, H., eds.) pp. 8-22, American Chemical Society Symposium Series 525, Washington, D.C.; Byers, J. A. (1995) in
Chemical Ecology of Insects
2 (Cardé, R. T., and Bell, W. J., eds.) pp. 154-213, Chapman and Hall, New York). In Grand fir (
Abies grandis
), increased formation of oleoresin monoterpenes, sesquiterpenes and diterpenes is induced by bark beetle attack (Lewinsohn, E., Gijzen, M., Savage, T. J., and Croteau, †R. (1991)
Plant Physiol.
96:38-43; Raffa, K. F., and Berryman, A. A. (1982)
Can. Etomol.
114:797-810; Lewinsohn, E., Gijzen, M., and Croteau, R. (1991)
Plant Physiol.
96:44-49), and this inducible defense response is mimicked by mechanically wounding sapling stems (Lewinsohn, E., Gijzen, M., Savage, T. J., and Croteau, R. (1991)
Plant Physiol.
96:38-43; Lewinsohn, E., Gijzen, M., and Croteau, R. (1991)
Plant Physiol.
96:44-49; Funk, C., Lewinsohn, E., Stofer Vogel, B., Steele C., and Croteau, R. (1994)
Plant Physiol.
106:999-1005). Therefore, Grand fir has been developed as a model system to study the biochemical and molecular genetic regulation of constitutive and inducible terpene biosynthesis in conifers (Steele, C., Lewinsohn, E., and Croteau, R. (1995)
Proc. Natl. Acad Sci. USA
92:4164-4168).
Most monoterpenes are derived from geranyl diphosphate, the ubiquitous C
10
intermediate of the isoprenoid pathway, by synthases which catalyze the divalent metal ion-dependent ionization (to 1,
FIG. 1
) and isomerization of this substrate to enzyme-bound linalyl diphosphate which, following rotation about C2-C3, undergoes a second ionization (to 2,
FIG. 1
) followed by cyclization to the &agr;-terpinyl cation, the first cyclic intermediate en route to both monocyclic and bicyclic products (Croteau, R., and Cane, D. E. (1985) Methods Enzymol. 110:383-405; Croteau, R. (1987)
Chem. Rev.
87:929-954) (FIG.
1
). Acyclic monoterpenes, such as myrcene, may arise by deprotonation of carbocations 1 or 2, whereas the isomerization step to linalyl diphosphate is required in the case of cyclic types, such as limonene and pinenes, which cannot be derived from geranyl diphosphate directly because of the geometric impediment of the trans-double bond at C2-C3 (Croteau, R., and Cane, D. E. (1985)
Methods Enzymol
110:383-405; Croteau, R. (1987)
Chem. Rev.
87:929-954). Many monoterpene synthases catalyze the formation of multiple products, including acyclic, monocyclic and bicyclic types, by variations on this basic mechanism (Gambliel, H., and Croteau, R. (1984)
J. Biol. Chem.
259:740-748; Croteau, R., Satterwhite, D. M., Cane, D. E., and Chang, C. C. (1988)
J. Biol. Chem.
263:10063-10071; Croteau, R., and Satterwhite, D. M. (1989)
J. Biol. Chem.
264:15309-15315). For example, (−)-limonene synthase, the principal monoterpene synthase of spearmint (
Mentha spicata
) and peppermint (
M. x piperita
), produces small amounts of myrcene, (−)-&agr;-pinene and (−)-&bgr;-pinene in addition to the monocyclic product (Rajaonarivony, J. I. M., Gershenzon, J., and Croteau, R. (1992)
Arch. Biochem. Biophys.
296:49-57; Colby, S. M., Alonso, W. R., Katahira, E. J., McGarvey, D. J., and Croteau, R. (1993)
J. Biol. Chem.
268:23016-23024. Conversely, six different inducible monoterpene synthase activities have been demonstrated in extracts of wounded Grand fir stem (Gijzen, M., Lewinsohi, E., and Croteau, R. (1991)
Arch. Biochem. Biophys.
289:267-273) indicating that formation of acyclic, monocyclic and bicyclic monoterpenes in this species involves several genes encoding distinct catalysts. The inducible (−)-pinene synthase has been purified (Lewinsohn, E., Gijzen, M., and Croteau, R. (1992)
Arch. Biochem. Biophys.
293:167-173), and isotopically sensitive branching experiments employed to demonstrate that this enzyme synthesizes both (−)-&agr;- and (−)-&bgr;-pinene (Wagschal, K., Savage, T. J., and Croteau, R. (1991)
Tetraheadon
47:5933-5944).
Deciphering the molecular genetic control of oleoresinosis and examining structure-function relationships among the monoterpene synthases of Grand fir requires isolation of the cDNA species encoding these key enzymes. Although a protein-based cloning strategy was recently employed to acquire a cDNA for the major wound-inducible diterpene synthase from Grand fir, abietadiene synthase (Funk, C., Lewinsohn, E., Stofer Vogel, B., Steele C., and Croteau, R. (1994)
Plant Physiol.
106:999-1005; LaFever, R. E., Stofer Vogel, B., and Croteau, R. (1994)
Arch. Biochem. Biophys.
313:139-149; Stofer Vogel, B., Wildung, M. R., Vogel, G., and Croteau, R. (1996)
J. Biol. Chem.
271:23262-23268), all attempts at the reverse genetic approach to cloning of Grand fir monoterpene synthases have failed (Steele, C., Lewinsohn, E., and Croteau, R. (1995)
Proc. Natl. Acad Sci. USA
92:4164-4168). As an alternative, a similarity-based PCR strategy was developed (Steele, C., Lewinsohn, E., and Croteau, R. (1995)
Proc. Natl. Acad Sci. USA
92:4164-4168) that employed sequence information from terpene synthases of angiosperm origin, namely a monoterpene synthase, (−)-4S-limonene synthase, from spearmint (
Mentha spicata,
Lamiaceae) (Colby, S. M., Alonso, W. R., Katahira, E. J., McGarvey, D. J., and Croteau, R. (1993)
J. Biol. Chem.
268:23016-23024), a sesquiterpene synthase, 5-epi-aristolochene synthase, from tobacco (
Nicotiana tabacum
, Solanaceae) (Facchini, P. J., and Chappell, J. (1992)
Proc. Natl. Acad. Sci. USA
89:11088-11092), and a diterpene synthase, casbene synthase,

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