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Abstract:
Grand fir (Abies grandis) is a useful model system for studying the biochemistry, molecular genetics, and regulation of defensive oleoresin formation in conifers,
a process involving both the constitutive accumulation
of resin (pitch) in specialized secretory structures
and the induced biosynthesis of monoterpenes
and sesquiterpenes (turpentine) and diterpene resin
acids (rosin) by nonspecialized cells at the site of injury.
A similarity-based cloning strategy, employing
primers designed to conserved regions of existing
monoterpene synthases and anticipated to amplify a
1000-bp fragment, unexpectedly yielded a 300-bp fragment
with sequence reminiscent of a terpenoid synthase.
Utilization of this amplicon as a hybridization
probe afforded four new, full-length cDNA species
from a wounded ®r stem cDNA library that appeared
to encode four distinct monoterpene synthases. Expression
in Escherichia coli, followed by enzyme assay
with geranyl diphosphate (C10), farnesyl diphosphate
(C15) and geranylgeranyl diphosphate (C20), and analysis
of the terpene products by chiral phase gas chromatography
and mass spectrometry con®rmed that
these sequences encoded four new monoterpene synthases,
including (2)-camphene synthase, (2)-b-phellandrene
synthase, terpinolene synthase, and an enzyme
that produces both (2)-limonene and (2)-apinene.
The deduced amino acid sequences indicated
these enzymes to be 618 to 637 residues in length (71 to
73 kDa) and to be translated as preproteins bearing an
amino-terminal plastid targeting sequence of 50±60
residues. cDNA truncation to delete the transit peptide
allowed functional expression of the ªpseudomature
º forms of these enzymes, which exhibited no
change in product outcome as a result of truncation.
Sequence comparison revealed that these new monoterpene
synthases from grand ®r are members of the
Tpsd gene subfamily and resemble sesquiterpene (C15)
synthases and diterpene (C20) synthases from conifers
more closely than mechanistically related monoterpene
synthases from angiosperm species. The availability
of a nearly complete set of constitutive and
inducible monoterpene synthases from grand ®r (now
numbering seven) will allow molecular dissection of
the resin-based defense response in this conifer species,
and detailed study of structure±function relationships
among this large and diverse family of catalysts,
all of which exploit the same stereochemistry in the
coupled isomerization±cyclization reaction.
