Terpenoid-based defenses in conifers: cDNA cloning, characterization, and functional expression of wound-inducible (E)-α-bisabolene synthase from grand fir (Abies grandis)

Terpenoid-based defenses in conifers: cDNA cloning, characterization, and functional expression of wound-inducible (E)-α-bisabolene synthase from grand fir (Abies grandis)

Institute of Biological Chemistry and Department of Biochemistry and Biophysics, Washington State University, Pullman, WA 99164-6340

Contributed by Rodney Croteau

Abstract

(E)-α-Bisabolene synthase is one of two wound-inducible sesquiterpene synthases of grand fir (Abies grandis), and the olefin product of this cyclization reaction is considered to be the precursor in Abies species of todomatuic acid, juvabione, and related insect juvenile hormone mimics. A cDNA encoding (E)-α-bisabolene synthase was isolated from a wound-induced grand fir stem library by a PCR-based strategy and was functionally expressed in Escherichia coli and shown to produce (E)-α-bisabolene as the sole product from farnesyl diphosphate. The expressed synthase has a deduced size of 93.8 kDa and a pI of 5.03, exhibits other properties typical of sesquiterpene synthases, and resembles in sequence other terpenoid synthases with the exception of a large amino-terminal insertion corresponding to Pro⁸¹–Val²⁹⁶. Biosynthetically prepared (E)-α-[³H]bisabolene was converted to todomatuic acid in induced grand fir cells, and the time course of appearance of bisabolene synthase mRNA was shown by Northern hybridization to lag behind that of mRNAs responsible for production of induced oleoresin monoterpenes. These results suggest that induced (E)-α-bisabolene biosynthesis constitutes part of a defense response targeted to insect herbivores, and possibly fungal pathogens, that is distinct from induced oleoresin monoterpene production.

Footnotes

↵ * Present address: Max-Planck-Institut für Chemische Ökologie, D-07745 Jena, Germany.
↵ † J.B. and J.C. contributed equally to this work.
↵ ‡ Present address: Universität Würzburg, Lehrstuhl für Botanik II, D-97082 Würzburg, Germany.
↵ § To whom reprint requests should be addressed. e-mail: croteau{at}mail.wsu.edu.
Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. AF006194 and AF006195).
ABBREVIATIONS:

FDP,

farnesyl diphosphate;

GDP,

geranyl diphosphate;

GGDP,

geranylgeranyl diphosphate;

RACE,

rapid amplification of cDNA ends