Chemical Ecology & Phytochemistry-2006

INDUCED CHEMICAL DEFENSES IN CONIFERS 13
distinguished. One is a group of homodimeric GPP synthases (sequences from
Citrus sinensis, Quercus robur, Arabidopsis thaliana, and PaIDS3). 44 Another
group of sequences represents the small subunit of the heterodimeric GPP synthases
from Antirrhinum majus and Mentha x piperita. 42,45 The corresponding large
subunits of these proteins nest separately within the main group of GGPP synthases,
an appropriate position, since these are reported to have GGPP synthase activity
when heterologously expressed along without their small subunit partners. 39,42 A
final group of GPP synthases is part of conifer homodimeric GPP and GGPP
synthases, including PaIDS1, 2 and 5. It appears that P. abies has a great number of
different types of GPP and GGPP synthases, perhaps appropriate for a plant that
makes such a variety of terpene metabolites. However, further speculation is
unwarranted until the catalytic function of these genes has been determined. For this
purpose, it is necessary to express them heterologously and assay the enzymatic
activity of the encoded proteins.
To this point, we have tested the expression of four of the six P. abies IDS
genes in E. coli by cloning them into expression vectors which produce proteins with
a fused His-tag to facilitate purification. Sequences for PaIPS1, PaIPS5, and
PaIPS6 were first truncated to remove putative transit peptides. The recombinant
proteins were extracted, purified on a Ni 2+ -agarose column (Fig. 1.7) and assayed
with IPP and each of the various allylic diphosphate substrates (Fig. 1.8). The
PaIDS4 protein was shown to make FPP with a small quantity of GPP. The proteins
designated PaIDS5 and PaIDS6 make solely GGPP, as might be expected from their
sequences, while curiously PaIDS1 makes GPP and GGPP in an approximate 2:1
ratio. There is as yet no precedent for an isoprenyl diphosphate synthase that makes
both GPP and GGPP in substantial amounts, but does not produce any FPP.
However, since P. abies terpene resin contains about equal amounts of GPP products
(monoterpenes) and GGPP products (diterpenes), the existence of an isoprenyl
diphosphate synthase that makes both in vivo is an intriguing possibility. Additional
study is underway to see if PaIDS1 makes both products in vivo. To learn more
about the role of these genes in the plant, we are also investigating their expression
pattern in various organs and tissues in relation to terpene formation. In addition, we
are developing a transformation system for P. abies and hope to use these genes to
try to manipulate terpene formation in transgenic saplings. Plants with altered
terpene profiles would supply ideal material for experiments to test the roles of
terpene resins against herbivores and pathogens.